Polynucleotides and methods for making plants resistant to fungal pathogens

ABSTRACT

This invention relates to polynucleotide sequences encoding genes that can confer resistance to the plant pathogen  Colletotrichum , which causes anthracnose stalk rot, leaf blight and top dieback in corn and other cereals. It further relates to plants and seeds of plants carrying chimeric genes comprising said polynucleotide sequences, which enhance or confer resistance to the plant pathogen  Colletotrichum , and processes of making said plants and seeds. The invention further presents sequences that can be used as molecular markers that in turn can be used to identify the region of interest in corn lines resulting from new crosses and to quickly and efficiently introgress the genes from corn lines carrying said genes into other corn lines that do not carry said genes, in order to make them resistant to  Colletotrichum  and resistant to stalk rot.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalApplication No. 60/944,209, filed on Jun. 15, 2007, which is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to compositions and methods useful in creating orenhancing pathogen-resistance in plants. Additionally, the inventionrelates to plants that have been genetically transformed with thecompositions of the invention.

BACKGROUND OF THE INVENTION

Colletotrichum graminicola (Ces.) (Cg), more commonly known asanthracnose, is the causative agent of anthracnose leaf blight,anthracnose stalk rot (ASR) and top dieback that affects Zea mays (L.),also known as maize or corn. It is the only known common stalk rot thatalso causes a leaf blight (Bergstrom, et al., (1999), Plant Disease,83:596-608, White, D. G. (1998), Compendium of Corn Diseases, pp. 1-78).It has been known to occur in the United States since 1855 and has beenreported in the Americas, Europe, Africa, Asia, and Australia (McGee, D.C. (1988), Maize Diseases: A Reference Source for Seed Technologists,APS Press, St. Paul, Minn.; White, (1998) supra; White, et al., (1979)Proc. Annu. Corn Sorghum Res Conf. (34^(th)), 1-15). In the UnitedStates alone, over 37.5 million acres are infested annually with averageyield losses of 6.6% nationwide (See FIG. 1). The yield losses are dueboth to low kernel weight in infected plants and “lodging,” that is, thefalling over of the plants due to weakness in the stalks caused by theinfection (Dodd, J., (1980), Plant Disease, 64:533-537). Lodged plantsare more difficult to harvest and are susceptible to other diseases.After infection, typically the upper portion of the stalk dies firstwhile the lower stalk is still green. Externally, infection can berecognized by blotchy black patches on the outer rind of the stalk,while internally the pith tissue is discolored or black in appearance.Inoculation occurs in a number of ways. Roots may grow through stalkdebris and become infected. This will become an increasing problem as“no till” methods of agriculture are more widely adopted due to theirenvironmental benefits. The fungus may also infect the stalks throughinsect damage and other wounds (White (1998) supra). Stalk infection maybe preceded by leaf infection causing leaf blight and providing inoculumfor stalk infection. There is controversy in the technical literature asto the number of different varieties or races of Cg present in nature.The pathogen is transmitted by wind or contaminated seed lots. Sporesremain viable for up to 2 years (McGee (1988) supra; Nicholson, et al.,(1980), Phytopathology, 70:255-261; Warren, H. L. (1977),Phytopathology, 67:160-162; Warren, et al., (1975), Phytopathology,65:620-623).

Farmers may combat infection by corn fungal diseases such as anthracnosethrough the use of fungicides, but these have environmental sideeffects, and require monitoring of fields and diagnostic techniques todetermine which fungus is causing the infection so that the correctfungicide can be used. Particularly with large field crops such as corn,this is difficult. The use of corn lines that carry genetic ortransgenic sources of resistance is more practical if the genesresponsible for resistance can be incorporated into elite, high yieldinggermplasm without reducing yield. Genetic sources of resistance to Cghave been described. There have been several maize lines identified thatcarry some level of resistance to Cg (White, et al. (1979) supra). Theseincluded A556, MP305, H21, SP288, CI88A, and FR16. A reciprocaltranslocation testcross analysis using A556 indicated that genescontrolling resistance to ASR lie on the long arms of chromosomes 1, 4,and 8 as well as both arms of chromosome 6 (Carson, M. L. (1981),Sources of inheritance of resistance to anthracnose stalk rot of corn.Ph.D. Thesis, University of Illinois, Urbana-Champaign). Introgressionof resistance derived from such lines is complex. Another inbred, LB31,was reported to carry a single dominant gene controlling resistance toASR but appears to be unstable, especially in the presence of Europeancorn borer infestation (Badu-Apraku et al., (1987) Phytopathology 77:957-959). The line MP305 was found to carry two dominant genes forresistance, one with a major effect and one with a minor effect (Carson(1981) supra). MP305 has been made available by the University ofMississippi through the National Plant Germplasm System (GRIN ID NSL250298) operated by the United States Department of Agriculture. SeeCompilation of North American Maize Breeding Germplasm, J. T. Gerdes etal., Crop Science Society of America, 1993. Seed of MP305 can beobtained through W. Paul Williams, Supervisory Research GeneticistUSDA-ARS, Corn Host Plant Resistance Research Unit, Box 9555, 340 DormanHall, Mississippi State, Miss. 39762.

It has been reported that there are two genetically separable (meaningthey behaved as separate genetic loci) genes linked on the long arm ofchromosome 4 that confer resistance to Cg (Toman, et al., (1993),Phytopathology, 83:981-986; Cowen, N et al. (1991) Maize GeneticsConference Abstracts 33). A significant resistance quantitative traitlocus (QTL) on chromosome 4 has also been reported (Jung, et al.,(1994), Theoretical and Applied Genetics, 89:413-418). Jung et al.(supra) reported that UMC15 could be used to select for the QTL onchromosome 4 in MP305, and suggested that the QTL is on a 12 cM regionof chromosome 4 between UMC15 and UMC66. In fact, as discussed in moredetail below, the region between UMC15 and UMC66 as reported on the IBM2neighbors 4 genetic map is approximately 129 cM, and selection for theQTL in the manner suggested by Jung et al. (1994, supra) would at bestselect a large chromosomal interval with considerable linkage drag andnegative phenotypic effect, and at worst, a double recombination couldoccur between the two markers resulting in a false positive selectionfor the Rcg1 locus. The region carrying the genes responsible for thephenotype conferred by the QTL on chromosome 4 will be referred toherein as the Rcg1 locus or the MP305 resistance locus; it has elsewherebeen referred to as the ASR locus.

Much work has been done on the mechanisms of disease resistance inplants in general. Some mechanisms of resistance are non-pathogenspecific in nature, or so-called “non-host resistance.” These may bebased on cell wall structure or similar protective mechanisms. However,while plants lack an immune system with circulating antibodies and theother attributes of a mammalian immune system, they do have othermechanisms to specifically protect against pathogens. The most importantand best studied of these are the plant disease resistance genes, or “R”genes. One of very many reviews of this resistance mechanism and the Rgenes can be found in Bekhadir et al., (2004), Current Opinion in PlantBiology 7:391-399. There are 5 recognized classes of R genes:intracellular proteins with a nucleotide-binding site (NBS) and aleucine-rich repeat (LRR); transmembrane proteins with an extracellularLRR domain (TM-LRR); transmembrane and extracellular LRR with acytoplasmic kinase domain (TM-CK-LRR); membrane signal anchored proteinwith a coiled-coil cytoplasmic domain (MSAP-CC); and membrane associatedkinases with an N-terminal myristylation site (MAK-N) (See, for example:Cohn, et al., (2001), Immunology, 13:55-62; Dangl, et al. (2001),Nature, 411:826-833).

Broglie et al. (U.S. patent application Ser. No. 11/397,153) described anovel R gene related to the NBS-LRR type designated Rcg1 found withinthe Rcg1 locus previously described by Jung et al. (supra). Theydescribed markers of use in breeding with this locus, chromosomalintervals which can endow corn plants with resistance, and transgenicplants containing the Rcg1 gene. The present invention improves on thework of Broglie et al. by providing a second NBS-LRR gene, differentfrom the Rcg1 gene and designated Rcg1b, that is required in combinationwith Rcg1 for resistance to Cg. The Rcg1b gene is physically (200-300kb) and genetically (less than 1 centimorgan) tightly linked to Rcg1 andrepresents a second component of the Rcg1 locus. The present inventionprovides sequences for Rcg1b, chimeric constructs combining Rcg1 andRcg1b, and methods and markers for breeding with the Rcg1 and Rcg1bgenes together (the Rcg1 locus).

SUMMARY OF THE INVENTION

Embodiments of this invention are based on the fine mapping, cloning andcharacterization of the genes responsible for the resistance phenotypefrom the line MP305, the introgression of a truncated chromosomalinterval with the MP305 resistance locus (Rcg1 locus) into other lineswith little or no linkage drag, the demonstration of the use of thosegenes as transgenes and the use of molecular markers to move the genesinto elite lines using breeding techniques.

Embodiments include an isolated polynucleotide comprising a nucleotidesequence encoding the Rcg1b polypeptide, which in conjunction with theRcg1 polypeptide, confers resistance to Colletotrichum, wherein the Rcg1polypeptide has an amino acid sequence of at least 50%, at least 75%, atleast 80%, at least 85%, at least 90%, and at least 95% identity, whencompared to SEQ ID NO:3 or the peptide encoded by the Rcg1 sequencedeposited with the Agricultural Research Service (ARS) CultureCollection on Feb. 22, 2006 as Patent Deposit No. NRRL B-30895, andfurther wherein the Rcg1b polypeptide has an amino acid sequence of atleast 50%, at least 75%, at least 80%, at least 85%, at least 90%, andat least 95% identity, when compared to SEQ ID NO:246 or the peptideencoded by the Rcg1b sequence deposited with the Agricultural ResearchService (ARS) Culture Collection on Jun. 26, 2007 as Patent Deposit No.NRRL B-50050 based on the Needleman-Wunsch alignment algorithm, or acomplement of the nucleotide sequence, wherein the complement and thenucleotide sequence consist of the same number of nucleotides and are100% complementary. Embodiments also include an isolated polynucleotidecomprising a nucleotide sequence encoding a polypeptide capable, inconjunction with the Rcg1 polypeptide, of conferring resistance toColletotrichum, wherein the polypeptide has an amino acid sequence of atleast 50%, at least 75%, at least 80%, at least 85%, at least 90%, andat least 95% identity, when compared to SEQ ID NO: 246.

Additional embodiments of the present invention include a vectorcomprising the polynucleotide that encodes a polypeptide of anembodiment of the present invention, such as SEQ ID NO: 3, SEQ ID NO:246, or the polynucleotide sequence of the plasmid deposited as PatentDeposit No. NRRL B-30895, or the polynucleotide sequence of the plasmiddeposited as Patent Deposit No. NRRL-B-50050, and a recombinant DNAconstruct comprising the polynucleotide of an embodiment of the presentinvention operably linked to at least one regulatory sequence.Additional embodiments of the present invention include vectorscomprising polynucleotides that encode both SEQ ID NO: 3 and SEQ ID NO:246. A plant cell, as well as a plant, each comprising a recombinant DNAconstruct of an embodiment of the present invention, and a seedcomprising a recombinant DNA construct of the embodiments are alsoembodied by the present invention.

The methods embodied by the present invention include 1) a method fortransforming a host cell, including a plant cell, comprisingtransforming the host cell with the polynucleotide of an embodiment ofthe invention, 2) a method for producing a plant comprising transforminga plant cell with the recombinant DNA construct of an embodiment of thepresent invention and regenerating a plant from the transformed plantcell, and 3) methods of conferring or enhancing resistance toColletotrichum and/or stalk rot, comprising transforming a plant withthe recombinant DNA construct of an embodiment of the present invention,thereby conferring and/or enhancing resistance to Colletotrichum orstalk rot.

Additional embodiments include methods of determining the presence orabsence of the polynucleotide of an embodiment of the present invention,or the Rcg1 locus, in a corn plant, comprising at least one of (a)isolating nucleic acid molecules from the corn plant and determining ifan Rcg1 gene is present or absent, and determining if an Rcg1b gene ispresent or absent, by amplifying sequences homologous to thepolynucleotide, (b) isolating nucleic acid molecules from the corn plantand performing a Southern hybridization, (c) isolating proteins from thecorn plant and performing a western blot using antibodies to the Rcg1protein and/or the Rcg1b protein, (d) isolating proteins from the cornplant and performing an ELISA assay using antibodies to the Rcg1 proteinand/or the Rcg1b protein, (e) demonstrating the presence of mRNAsequences derived from the Rcg1 and/or Rcg1b mRNA transcript and uniqueto Rcg1 and/or Rcg1b, or (f) demonstrating the presence of newlyconferred or enhanced resistance to Colletotrichum infection through aphenotypic assay, thereby determining the presence of thepolynucleotides or the Rcg1 locus in the corn plant.

Methods of altering the level of expression of a protein capable ofconferring resistance to Colletotrichum or stalk rot in a plant or plantcell comprising (a) transforming a plant cell with the recombinant DNAconstruct of an embodiment of the present invention and (b) growing thetransformed plant cell under conditions that are suitable for expressionof the recombinant DNA construct wherein expression of the recombinantDNA construct results in production of altered levels of a proteincapable of conferring resistance to Colletotrichum or stalk rot in thetransformed host are also embodied by the present invention.

An additional method embodied by the present invention is a method ofconferring or enhancing resistance to Colletotrichum and/or stalk rot ina corn plant, comprising (a) crossing a first corn plant lacking theRcg1 locus with a second corn plant containing the Rcg1 locus to producea segregating population, (b) screening the segregating population for amember containing the Rcg1 locus with a first nucleic acid, notincluding UMC15a or UMC66, capable of hybridizing with a second nucleicacid linked to or located within the Rcg1 locus, and (c) selecting themember for further crossing and selection.

Methods of enhancing resistance to Colletotrichum and/or stalk rot, orintrogressing Colletotrichum and/or stalk rot resistance into a cornplant, comprising performing marker assisted selection of the corn plantwith a nucleic acid marker, wherein the nucleic acid marker specificallyhybridizes with a nucleic acid molecule having a first nucleic acidsequence that is linked to a second nucleic acid sequence that islocated on the Rcg1 locus of MP305 and selecting the corn plant based onthe marker assisted selection are also embodiments of the presentinvention. Specific FLP, MZA and Rcg1-specific SNP markers disclosedherein are further aspects of the invention.

Another embodiment of the invention is a process of identifying a cornplant that displays newly conferred or enhanced resistance toColletotrichum infection by detecting alleles of at least one marker onor within the Rcg1b gene, and optionally by detecting a second marker onor within the Rcg1 gene.

Further embodiments include processes for identifying corn plants thatdisplay newly conferred or enhanced resistance to Colletotrichum bydetecting alleles of at least 2 markers in the corn plant, wherein atleast one of the markers is on or within the chromosomal interval belowUMC2041 and above the Rcg1b gene, and at least one of the markers is onor within the interval below the Rcg1 gene and above UMC2200. Similarembodiments encompassed by this process include at least one of themarkers being on or within the chromosomal interval below UMC1086 andabove the Rcg1b gene, on or within the chromosomal interval belowUMC2285 and above the Rcg1b gene, and at least one of the markers is onor within the interval below the Rcg1 gene and above UMC2200, on orwithin the interval below the Rcg1 gene and above UMC2187, or on orwithin the interval below the Rcg1 gene and above UMC15a.

Further embodiments include processes for identifying corn plants thatdisplay newly conferred or enhanced resistance to Colletotrichum bydetecting alleles of at least 2 markers in the corn plant, wherein atleast one of the markers on or within the chromosomal interval belowUMC2041 and above the Rcg1b gene is selected from the markers listed inTable 16, and at least one of the markers on or within the intervalbelow the Rcg1 gene and above UMC2200 is also selected from the markerslisted in Table 16. Embodiments include processes for identifying cornplants that display newly conferred or enhanced resistance toColletotrichum by selecting for at least four markers or at least six,wherein at least two or three of the markers are on or within thechromosomal interval below UMC2041 and above the Rcg1b gene, and atleast two or three of the markers are on or within the interval belowthe Rcg1 gene and above UMC2200. Additional embodiments include thissame process when the two or three markers on or within the chromosomalinterval below UMC2041 and above the Rcg1b gene, as well as the two orthree markers on or within the interval below the Rcg1 gene and aboveUMC2200, are selected from those listed in Table 16. Another embodimentof this process includes detecting allele 7 at MZA112, detecting allele2 at MZA2591, or detecting allele 8 at MZA3434. Corn plants and seedsproduced by the embodied processes are also embodiments of theinvention, including those corn plants which do not comprise the samealleles as MP305 at or above UMC2041, or at or below UMC2200 at the locishown in Table 16.

Other embodiments include processes for identifying corn plants thatdisplay newly conferred or enhanced resistance to Colletotrichum bydetecting alleles of at least 2 markers in the corn plant, wherein atleast one of the markers is on or within the chromosomal interval belowUMC2041 and above the Rcg1b gene, and at least one of the markers is onor within the interval below the Rcg1 gene and above UMC2200, and wherethe process detects the presence or absence of at least one markerlocated within the Rcg1 gene or the Rcg1b gene. A further suchembodiment includes a modification of this process in which four markersare selected for, in which two of the markers are within the chromosomalinterval below UMC2285 and above the Rcg1b gene, and at least two of themarkers are within the interval below the Rcg1 gene and above UMC15a. Afurther embodiment of this process includes the Rcg1 gene and the Rcg1bgene having been introgressed from a donor corn plant, including MP305or DE811ASR(BC5), into a recipient corn plant to produce an introgressedcorn plant. This process also includes the instance when theintrogressed corn plant is selected for a recombination event below theRcg1 gene and above UMC15a, so that the introgressed corn plant retainsa first MP305 derived chromosomal interval below the Rcg1 gene and aboveUMC15a, and does not retain a second MP305 derived chromosomal intervalat and below UMC15a. Corn plants and seeds produced by these processesare also embodiments of the invention. Introgressed corn plants embodiedby the invention include those that are Rcg1 locus conversions of PH705,PH5W4, PH51K or PH87P, or progeny thereof.

A further embodiment of the invention is a process of identifying a cornplant that displays enhanced resistance to Colletotrichum infection, bydetecting in the corn plant the presence or absence of at least onemarker at the Rcg1 locus, and selecting the corn plant in which the atleast one marker is present. Embodiments include when at least onemarker is on or within SEQ ID NOs: 137, 255, 256, 257, 258, or 266 andalso when at least one marker is on or within the Rcg1 or Rcg1b codingsequence, or located on or within the polynucleotide set forth in SEQ IDNO: 1 or SEQ ID NO: 245.

Introgressed corn plants embodied by the invention include those thatare Rcg1 locus conversions of PH705, PH5W4, PH51K or PH87P, or progenythereof. Such embodiments include corn seed comprising a first MP305derived chromosomal interval defined by BNLG2162 and UMC1051, and notcomprising a second MP305 derived chromosomal interval above UMC2041 orbelow UMC1051, and when the corn seed comprises at least one of the Rcg1and the Rcg1b genes and, when grown, produces a corn plant that exhibitsresistance to Colletotrichum infection. Seed of the embodiments alsoincludes corn seed comprising a first MP305 derived chromosomal intervalbetween, but not including, UMC2285 and UMC15a, and not comprising asecond MP305 derived chromosomal interval at or above UMC2285 or at orbelow UMC15a, and furthermore such corn seed which comprises the Rcg1and the Rcg1b genes and, when grown, produces a corn plant that exhibitsresistance to Colletotrichum infection. Corn plants and plant cellsproduced from this seed are also included in the embodiments of theinvention.

Progeny seed that is an Rcg1 locus conversion of PH705, PH5W4, PH51K orPH87P, or a progeny thereof is also embodied in the invention, as areprogeny seed that comprise at least two or more of allele 7 at MZA11123,allele 2 at MZA2591, or allele 8 at MZA3434. Further embodiments includeprogeny seed which comprise a cytosine nucleotide at MZA2591.32, athymine nucleotide at MZA2591.35, and a cytosine nucleotide atMZA3434.17.

Embodiments also include genetic markers on or within SEQ ID NOs: 140through 146 for MZA3434, MZA2591, MZA11123, MZA15842, MZA1851, MZA8761and MZA11455, respectively. Other embodiments include genetic markerslocated on or in the Rcg1 locus or the Rcg1 gene or the Rcg1b gene,including those located on SEQ ID NO: 137 or on any of SEQ ID NOs: 255,256, 257, 258 and 266. Embodied markers also include those located onSEQ ID NO: 1 or SEQ ID NO: 245.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a map of the United States showing the severity of anthracnosestalk rot infestation by county for 2002.

FIG. 2 (a, b, c) is an alignment of a polypeptide sequence of theembodiments (SEQ ID NO: 3) comparing it to other known NBS-LRRpolypeptides.

FIG. 3 is a graph produced by Windows QTL Cartographer software showinga statistical analysis of the chance (Y axis) that the locus responsiblefor the Cg resistance phenotype is located at a particular positionalong the chromosome (X axis) as defined by FLP markers.

FIG. 4 is an electrophoresis gel blot of aliquots of RT-PCR reactionswhich reveals the presence of a 260 bp band present in the samplesderived from both infected and uninfected resistant plants but absentfrom susceptible samples. RT-PCR fragments were obtained from 12.5 ngtotal RNA from DE811 and DE811 ASR stalk tissue. cDNA obtained byreverse transcription was amplified using Rcg1 specific primers and 18SrRNA primers as an internal standard.

FIG. 5 is a schematic diagram of the Mu-tagging strategy used tovalidate the Rcg1 gene.

FIG. 6 is the gene structure of Rcg1 showing the location of fourdifferent mutator insertion sites.

FIG. 7( a-b) is a series of genetic map images with increasingresolution of the map of the region near the Rcg1 locus. Map distancesfor 7(a) for the map labelled “A” are in cM and in relation to the IBM2Neighbors 4 genetic map. Map distances for 7(b) for the map labelled “B”were developed using 184 individuals from the BC7 population, and mapdistances for 7(b) for the map labelled “C” were developed using 1060individuals from the BC7 population. Genetic mapping in the BC7population increased the map resolution greater than 10-fold, whencompared with the published map. The location of the markers shown tothe right of each map is based on extrapolation of their location on thephysical map.

FIG. 8( a-b) is a genetic map image showing the chromosomal intervalwith the Rcg1 locus in DE811ASR (BC3), the reduced size of thechromosomal interval with the Rcg1 locus obtained in DE811ASR (BC5) andthe further reduced size of the chromosomal interval in inbreds obtainedby initially using DE811ASR (BC5) as a donor source. For all markers,the map distances shown were reported on the IBM2 neighbors map publiclyavailable on the Maize GDB, apart from for MZA15842, FLP27 and FLP56 forwhich map positions were extrapolated using regression analysis relativeto the high resolution maps in FIG. 7( b), maps B and C, using thepositions of UMC2285, PHI093 and CSU166a which were common to both maps.

FIGS. 9( a-b). FIG. 9( a) shows the alignment of the non-colinear regionfrom DE811ASR (BC5) relative to B73 and Mo17. The BAC sizes in FIG. 9(a) are estimates. FIG. 9( b) shows a portion of the non-colinear regionas set forth in SEQ ID NO: 137 on which Rcg1 resides, including therepetitive regions therein, as well as the Rcg1 exons 1 and 2.

FIG. 10 (a-b) show distributions of average leaf lesion size indifferent individual plants at 15 days after inoculation with Cg in theDE811ASR(BC5) and DE811 lines, respectively.

FIG. 11 shows a comparison of average leaf lesion size on plants ofDE811 and DE811ASR(BC5) infected with Cg at 7 and 15 days afterinoculation.

FIG. 12 shows the average severity of disease four to five weeks afterinoculation with Cg in stalks of hybrids derived from crossingDE811ASR(BC5) and DE811 to the line indicated.

FIG. 13 shows the improvement in yield at maturity after inoculationwith Cg in hybrids derived from crossing DE811ASR(BC5) to the lineindicated when compared to the yield of hybrids derived from crossingDE811 to the line indicated.

FIG. 14 shows the severity of disease at 5 different locations caused byCg in stalks of inbred lines derived from DE811ASR(BC5) or MP305 four tofive weeks after inoculation. Differences between the lines which werepositive and negative for the Rcg1 gene are statistically significant ata P value of less than 0.05.

FIG. 15 shows disease progression in representative stalks from inbredPH705 lines which are positive and negative for Rcg1.

FIG. 16 shows disease progression in representative stalks from inbredPH87P lines which are positive and negative for Rcg1.

FIG. 17 shows the severity of disease four to five weeks afterinoculation at 5 different locations caused by Cg in stalks of hybridsderived from crossing DE811ASR(BC5) to the line indicated. Differencesbetween the lines which were positive and negative for the Rcg1 gene arestatistically significant at a P value of less than 0.05, except forlocation 5.

FIG. 18 shows disease progression in representative stalks from hybridscreated from PH4CV and PH705 lines which are positive and negative forRcg1.

FIG. 19 shows disease progression in representative stalks from hybridscreated from PH705 and PH87P lines which are positive and negative forRcg1.

FIG. 20 shows the method of scoring for disease severity in corn stalks.The stalks are given a score, designated antgr75, which represents thenumber of internodes (up to 5, including the inoculated internode) thatare more than 75% discolored. This results in a score ranging from 0 to5, with 0 indicating less than 75% discoloration in the inoculatedinternode, and 5 indicating 75% or more discoloration of the first fiveinternodes, including the inoculated internode.

FIG. 21 shows a contig on the B73 physical map that is homologous to theregion into which the Rcg1 non-colinear region containing DE811ASR (BC5)is inserted, which demonstrates that many B73 derived bacterialartificial chromosomes (BACs) are available in the region of interestfrom which sequence information can be obtained.

FIG. 22 shows the alignment of the genetic map containing MZA and publicmarkers with the physical maps of Mo17 and B73. The genetic mapdistances were developed by using 1060 individuals from the BC7 mappingpopulation. An analysis of a Mo17 BAC library also showed the Rcg1 locusto be non-colinear with the corresponding region of Mo17. The locationof the markers shown by dotted lines to the B73 map are extrapolationsfrom the Mo17 physical map location. The location of the markers shownby dotted lines to the Mo17 map are extrapolations from the B73 physicalmap location.

FIG. 23 shows the oligos for the Rcg1 hybridization markers designed foruse with INVADER™ detection system reactions.

FIG. 24 shows the oligos for the Rcg1 hybridization markers designed foruse with TAQMAN® detection system reactions.

FIG. 25 shows the results of a northern blot obtained from approximately1.5 mg of polyA-enriched RNA isolated from resistant and susceptibleplants 0, 3, 6, 9, and 13 days post inoculation (dpi). The membrane wasprobed with a random primer labeled 420 bp Rcg1 fragment. Resistanttissue is from DE811ASR(BC5) and susceptible tissue is from DE811.

FIG. 26 shows that PCR amplification using Rcg1 specific primer pairsonly amplifies in the resistant line DE811ASR(BC5) and donor parentMP305, but not in susceptible line DE811, with the exception ofFLP110F-R, which amplifies the coiled coil-nucleotide binding siteregion, which is highly conserved, and thus amplifies a region elsewherein the genome that is not Rcg1 in the DE811 line. A 100 bp ladder wasused for fragment sizing.

FIG. 27 shows a series of leaf sheaths of V7-9 maize plants which wereinoculated with a suspension of Cg conidia following wounding of tissue.These results are further discussed in Example 8. In the absence of theRcg1 transgene (top row), leaf sheaths of plants containing the Rcg1gene display large, expanding necrotic lesions. In contrast, when theRcg1 transgene (in this case driven by its own promoter) is present(bottom row), an ASR resistant phenotype is restored and necrosis isconfined/restricted to the site of wounding and fungal inoculation.

FIG. 28 shows the results of RT-PCR performed with the ETJ115-ETJ116primer pair. This data clearly shows that the NBS-LRR gene of BAC clone191 supercontig H is expressed exclusively in DE811ASR plants thatcontain the MP305 introgressed region, as discussed in Example 9.

FIG. 29 shows the predicted domain structure of the Rcg1b gene, asdiscussed in Example 9.

FIG. 30 shows the result of a northern blot analysis which allowed anindependent confirmation of Rcg1b expression and provided an estimate oftranscript size. Northern blots of polyA+ RNA (0.7 mg) from resistantand susceptible plants (0, 6, 13 DPI) showed the presence of a 4.75 kbtranscript band, present only in the resistant samples that hybridizedspecifically with probes prepared from the LRR and the kinase region ofthe gene, as discussed in Example 9.

FIGS. 31 a through 31 g provide an alignment of the Rcg1b peptide (SEQID NO: 246) of the embodiments with several NBS-LRR proteins from barleyand rice (SEQ ID NOs: 247-251), showing regions of homology.

FIGS. 32 a and 32 b provide an alignment of amino acids 1036-1399 of SEQID NO: 246, which are separately set forth as SEQ ID NO: 256, withseveral putative protein kinases (SEQ ID NOs: 252-254) identified in aBLAST®P homology search.

FIG. 33 summarizes the leaf sheath infection data for Rcg1 KO plants inthe absence and in the presence of the native Rcg1 transgene, asdiscussed in Example 8.

FIG. 34 shows the results of leaf sheath assays from plants transformedwith Construct E as described in Example 10. A clear pattern of Cginfection is visible.

FIG. 35 shows the results of leaf sheath assays from plants transformedwith Construct C as described in Example 10. A clear pattern of Cginfection is visible.

FIG. 36 shows the results of leaf sheath assays from plants transformedwith Construct D as described in Example 10. The leaves clearly showinhibition of Cg infection as a result of the simultaneous presence andexpression of both the Rcg1 and Rcg1b genes.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention provide compositions and methods(or processes) directed to inducing pathogen resistance, particularlyfungal resistance, in plants. The compositions are novel nucleotide andamino acid sequences that confer or enhance resistance to plant fungalpathogens. Specifically, certain embodiments provide polypeptides havingthe amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 246,and variants and fragments thereof. Isolated nucleic acid molecules, andvariants and fragments thereof, comprising nucleotide sequences thatencode the amino acid sequences shown in SEQ ID NO: 3 and SEQ ID NO: 246are further provided.

Nucleotide sequences that encode the polypeptide of SEQ ID NO: 3 are setforth in SEQ ID NOs: 1 and 4. Nucleotide sequences that encode thepolypeptide of SEQ ID NO: 246 are set forth in SEQ ID NOs: 255-258, aswell as 246 and 266. The genomic sequence of the Rcg1b gene and itsflanking regions is provided in SEQ ID NOs: 255-258, or in SEQ ID NO:266, which are discussed in more detail in Example 9. The cDNA sequencethat encodes the polypeptide of SEQ ID NO: 246 is set forth in SEQ IDNO: 245. Plants, plant cells, seeds, and microorganisms comprising anucleotide sequence that encodes a polypeptide of the embodiments arealso disclosed herein.

A deposit of the Rcg1 nucleic acid molecule was made on Feb. 22, 2006with the Agricultural Research Service (ARS) Culture Collection, housedin the Microbial Genomics and Bioprocessing Research Unit of theNational Center for Agricultural Utilization Research (NCAUR), under theBudapest Treaty provisions. The deposit was given the followingaccession number: NRRL B-30895. The address of NCAUR is 1815 N.University Street, Peoria, Ill., 61604. This deposit will be maintainedunder the terms of the Budapest Treaty on the International Recognitionof the Deposit of Microorganisms for the Purposes of Patent Procedure.This deposit was made merely as a convenience for those of skill in theart and is not an admission that a deposit is required under 35 U.S.C.§112. The deposit will irrevocably and without restriction or conditionbe available to the public upon issuance of a patent. However, it shouldbe understood that the availability of a deposit does not constitute alicense to practice the subject invention in derogation of patent rightsgranted by government action.

A sample of 2500 seeds of DE811ASR (BC5) were deposited in the AmericanType Culture Collection (ATCC), 10801 University Blvd., Manassas, Va.20110-2209, USA on Mar. 13, 2006 and assigned Deposit No. PTO-7434.Access to this deposit will be available during the pendency of theapplication to the Commissioner of Patents and Trademarks, personsdetermined by the Commissioner to be entitled thereto upon request, andcorresponding officials in foreign patent offices in which this patentapplication is filed. This deposit will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure. The deposit willirrevocably and without restriction or condition be available to thepublic upon issuance of a patent. However, it should be understood thatthe availability of the deposit does not constitute a license topractice the subject invention or methods in derogation of patentrights.

A deposit of the Rcg1b nucleic acid molecule was made on Jun. 26, 2007with the Agricultural Research Service (ARS) Culture Collection, housedin the Microbial Genomics and Bioprocessing Research Unit of theNational Center for Agricultural Utilization Research (NCAUR), under theBudapest Treaty provisions. The deposit was given the followingaccession number: NRRL B-50050. The address of NCAUR is 1815N.University Street, Peoria, Ill., 61604. This deposit will be maintainedunder the terms of the Budapest Treaty on the International Recognitionof the Deposit of Microorganisms for the Purposes of Patent Procedure.This deposit was made merely as a convenience for those of skill in theart and is not an admission that a deposit is required under 35 U.S.C.§112. The deposit will irrevocably and without restriction or conditionbe available to the public upon issuance of a patent. However, it shouldbe understood that the availability of a deposit does not constitute alicense to practice the subject invention in derogation of patent rightsgranted by government action.

The two full length polypeptides of the embodiments (SEQ ID NOs: 3 and246) share varying degrees of homology with known polypeptides of theNBS-LRR family. In particular, Rcg1 (SEQ ID NO: 3) shares homology withNBS-LRR proteins isolated from Oryza sativa (Accession Nos. NP_(—)910480(SEQ ID NO: 14), NP_(—)910482 (SEQ ID NO: 16), NP_(—)921091 (SEQ ID NO:17) and NP_(—)910483 (SEQ ID NO: 15)) and Hordeum vulgare (Accession No.AAG37354 (SEQ ID NO: 18); Zhou et al., (2001) Plant Cell 13:337-350).FIG. 2 provides an alignment of the amino acid sequence set forth in SEQID NO: 3 with the O. sativa and H. vulgare proteins (SEQ ID NOs: 14-18).Amino acid alignments using the GAP program to compare SEQ ID NO: 3 tothe various rice and barley sequences identified above indicated thatSEQ ID NO:3 shares approximately 42.3% sequence similarity with the O.sativa protein NP_(—)910480 (SEQ ID NO: 14), 41.7% sequence similaritywith the O. sativa protein NP_(—)910482 (SEQ ID NO: 16), 56.9%similarity with the O. sativa protein NP_(—)921091 (SEQ ID NO: 17) and42.1% sequence similarity with the O. sativa protein NP_(—)910483 (SEQID NO: 15). Furthermore, SEQ ID NO: 3 shares approximately 42.8%sequence similarity with the H. vulgare protein AAG37354 (SEQ ID NO:18).

Similar analysis was conducted for the Rcg1b peptide (SEQ ID NO: 246),which was found to share homology with NBS-LRR proteins isolated fromOryza sativa (Accession Nos. ABA95308 (SEQ ID NO: 250), ABG66042 (SEQ IDNO: 251), ABA92208 (SEQ ID NO: 247) and ABG66044 (SEQ ID NO: 248)) andHordeum vulgare (Accession No. AAM22820 (SEQ ID NO: 249)). FIG. 31 athrough 31 g provides an alignment of the amino acid sequence set forthin SEQ ID NO: 246 with the O. sativa and H. vulgare proteins with theaccession numbers provided above (SEQ ID NOs: 247-251). Amino acidalignments using the ClustalW pair-wise alignments (Gap openingpenalty=10, Gap extension penalty=0.1) indicate that SEQ ID NO: 246shares approximately 37.1% sequence identity with the O. sativa proteinABA92208 (SEQ ID NO: 247), 28.2% sequence identity with the O. sativaprotein ABG66044 (SEQ ID NO: 248), 26.6% identity with the O. sativaprotein ABA95308 (SEQ ID NO: 250) and 31.1% sequence similarity with theO. sativa protein ABG66042 (SEQ ID NO: 251). Furthermore, SEQ ID NO: 3shares approximately 25.6% sequence similarity with the H. vulgareprotein AAM22820 (SEQ ID NO: 249).

The NBS-LRR group of R-genes is the largest class of R-genes discoveredto date. In Arabidopsis thaliana, over 150 are predicted to be presentin the genome (Meyers, et al., (2003), Plant Cell, 15:809-834; Monosi,et al., (2004), Theoretical and Applied Genetics, 109:1434-1447), whilein rice, approximately 500 NBS-LRR genes have been predicted (Monosi,(2004) supra). The NBS-LRR class of R genes is comprised of twosubclasses. Class 1 NBS-LRR genes contain a TIR-Toll/Interleukin-1 likedomain at their N′ terminus; which to date have only been found indicots (Meyers, (2003) supra; Monosi, (2004) supra). The second class ofNBS-LRR contain either a coiled-coil domain or an (nt) domain at their Nterminus (Bai, et al. (2002) Genome Research, 12:1871-1884; Monosi,(2004) supra; Pan, et al., (2000), Journal of Molecular Evolution,50:203-213). Class 2 NBS-LRR have been found in both dicot and monocotspecies. (Bai, (2002) supra; Meyers, (2003) supra; Monosi, (2004) supra;Pan, (2000) supra).

The NBS domain of the gene appears to have a role in signaling in plantdefense mechanisms (van der Biezen, et al., (1998), Current Biology: CB,8:R226-R227). The LRR region appears to be the region that interactswith the pathogen AVR products (Michelmore, et al., (1998), Genome Res.,8:1113-1130; Meyers, (2003) supra). This LRR region in comparison withthe NBS domain is under a much greater selection pressure to diversify(Michelmore, (1998) supra; Meyers, (2003) supra; Palomino, et al.,(2002), Genome Research, 12:1305-1315). LRR domains are found in othercontexts as well; these 20-29-residue motifs are present in tandemarrays in a number of proteins with diverse functions, such ashormone-receptor interactions, enzyme inhibition, cell adhesion andcellular trafficking. A number of recent studies revealed theinvolvement of LRR proteins in early mammalian development, neuraldevelopment, cell polarization, regulation of gene expression andapoptosis signaling.

The gene of the embodiments is clearly related to the NBS-LRR of theclass 2 family, but does not completely fit the classical mold. Theamino end has homology to so-called nucleotide binding sites (NBS).There is a leucine rich region as well, located, as expected, downstreamof the NBS. However, unlike previously studied NBS-LRR proteins, theleucine rich region lacks the systematic repetitive nature found in moreclassical LRR domains, much less consistently following the typical Lxxrepeat pattern and in particular having no instances of the consensussequences described by Wang et al. ((1999) Plant J. 19:55-64; seeespecially, FIG. 5) or Bryan et al. ((2000), Plant Cell 12:2033-2045;see especially, FIG. 3). Rcg1b, in addition to the NBS and LRR domains,contains a protein kinase domain.

As the LRR region is the receptor portion of an NBS-LRR, when a new LRRsuch as those of this disclosure are found, the range of its activity,that is, the range of pathogens to which it will respond, is notimmediately obvious from the sequence. The genes of the embodiments wereisolated on the basis of the Cg resistance phenotype, and therefore thenovel LRR responds to Cg. However, it is not excluded that they respondto other pathogens not tested in the work done heretofore.

The nucleic acids and polypeptides of the embodiments find use inmethods for conferring or enhancing fungal resistance to a plant.Accordingly, the compositions and methods disclosed herein are useful inprotecting plants from fungal pathogens. “Pathogen resistance,” “fungalresistance,” and “disease resistance” are intended to mean that theplant avoids the disease symptoms that are the outcome of plant-pathogeninteractions. That is, pathogens are prevented from causing plantdiseases and the associated disease symptoms, or alternatively, thedisease symptoms caused by the pathogen are minimized or lessened, suchas, for example, the reduction of stress and associated yield loss. Oneof skill in the art will appreciate that the compositions and methodsdisclosed herein can be used with other compositions and methodsavailable in the art for protecting plants from pathogen attack.

Hence, the methods of the embodiments can be utilized to protect plantsfrom disease, particularly those diseases that are caused by plantfungal pathogens. As used herein, “fungal resistance” refers to enhancedresistance or tolerance to a fungal pathogen when compared to that of awild type plant. Effects may vary from a slight increase in tolerance tothe effects of the fungal pathogen (e.g., partial inhibition) to totalresistance such that the plant is unaffected by the presence of thefungal pathogen. An increased level of resistance against a particularfungal pathogen or against a wider spectrum of fungal pathogensconstitutes “enhanced” or improved fungal resistance. The embodiments ofthe invention also will enhance or improve fungal plant pathogenresistance, such that the resistance of the plant to a fungal pathogenor pathogens will increase. The term “enhance” refers to improve,increase, amplify, multiply, elevate, raise, and the like. Herein,plants of the invention are described as being resistant to infection byCg or having ‘enhanced resistance’ to infection by Cg as a result of theRcg1 locus of the invention. Accordingly, they typically exhibitincreased resistance to the disease when compared to equivalent plantsthat are susceptible to infection by Cg because they lack the Rcg1locus. For example, using the scoring system described in Example 11(also see FIG. 20), they typically exhibit a one point, two point orthree point or more decrease in the infection score, or even a reductionof the score to 1 or 0, when compared to equivalent plants that aresusceptible to infection by Cg because they lack the Rcg1 locus.

In particular aspects, methods for conferring or enhancing fungalresistance in a plant comprise introducing into a plant at least oneexpression cassette, wherein the expression cassette comprises anucleotide sequence encoding the antifungal polypeptides of theembodiments each operably linked to a promoter that drives expression inthe plant. The plant expresses the two polypeptides, thereby conferringfungal resistance upon the plant, or improving the plant's inherentlevel of resistance. In particular embodiments, the gene confersresistance to the fungal pathogen, Cg. The skilled artisan could, ifdesired, create two separate transgenic plants each expressing one ofthe two polypeptides and then cross the two plants, selecting for plantsthat express both polypeptides.

Expression of an antifungal polypeptide of the embodiments may betargeted to specific plant tissues where pathogen resistance isparticularly important, such as, for example, the leaves, roots, stalks,or vascular tissues. Such tissue-preferred expression may beaccomplished by root-preferred, leaf-preferred, vasculartissue-preferred, stalk-preferred, or seed-preferred promoters.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues (e.g., peptide nucleic acids) having the essential nature ofnatural nucleotides in that they hybridize to single-stranded nucleicacids in a manner similar to naturally occurring nucleotides.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. Polypeptides of the embodiments can be produced either from anucleic acid disclosed herein, or by the use of standard molecularbiology techniques. For example, a truncated protein of the embodimentscan be produced by expression of a recombinant nucleic acid of theembodiments in an appropriate host cell, or alternatively by acombination of ex vivo procedures, such as protease digestion andpurification.

As used herein, the terms “encoding” or “encoded” when used in thecontext of a specified nucleic acid mean that the nucleic acid comprisesthe requisite information to direct translation of the nucleotidesequence into a specified protein. The information by which a protein isencoded is specified by the use of codons. A nucleic acid encoding aprotein may comprise non-translated sequences (e.g., introns) withintranslated regions of the nucleic acid or may lack such interveningnon-translated sequences (e.g., as in cDNA).

The embodiments of the invention encompass isolated or substantiallypurified polynucleotide or protein compositions. An “isolated” or“purified” polynucleotide or protein, or biologically active portionthereof, is substantially or essentially free from components thatnormally accompany or interact with the polynucleotide or protein asfound in its naturally occurring environment. Thus, an isolated orpurified polynucleotide or protein is substantially free of othercellular material, or culture medium when produced by recombinanttechniques (e.g. PCR amplification), or substantially free of chemicalprecursors or other chemicals when chemically synthesized. Optimally, an“isolated” polynucleotide is free of sequences (for example, proteinencoding sequences) that naturally flank the polynucleotide (i.e.,sequences located at the 5′ and 3′ ends of the polynucleotide) in thegenomic DNA of the organism from which the polynucleotide is derived.For example, in various embodiments, the isolated polynucleotide cancontain less than about 5 kb, about 4 kb, about 3 kb, about 2 kb, about1 kb, about 0.5 kb, or about 0.1 kb of nucleotide sequence thatnaturally flank the polynucleotide in genomic DNA of the cell from whichthe polynucleotide is derived. A protein that is substantially free ofcellular material includes preparations of protein having less thanabout 30%, about 20%, about 10%, about 5%, or about 1% (by dry weight)of contaminating protein. When the protein of the embodiments, or abiologically active portion thereof, is recombinantly produced,optimally culture medium represents less than about 30%, about 20%,about 10%, about 5%, or about 1% (by dry weight) of chemical precursorsor non-protein-of-interest chemicals.

Fragments and variants of the disclosed nucleotide sequences andproteins encoded thereby are also encompassed by the embodiments.“Fragment” is intended to mean a portion of the nucleotide sequence or aportion of the amino acid sequence and hence protein encoded thereby.Fragments of a nucleotide sequence may encode protein fragments thatretain the biological activity of the native protein and hence have theability to confer fungal resistance upon a plant. Alternatively,fragments of a nucleotide sequence that are useful as hybridizationprobes do not necessarily encode fragment proteins retaining biologicalactivity. Thus, fragments of a nucleotide sequence may range from atleast about 15 nucleotides, about 50 nucleotides, about 100 nucleotides,and up to the full-length nucleotide sequence encoding the polypeptidesof the embodiments.

A fragment of a nucleotide sequence that encodes a biologically activeportion of a polypeptide of the embodiments will encode at least about15, about 25, about 30, about 40, or about 50 contiguous amino acids, orup to the total number of amino acids present in a full-lengthpolypeptide of the embodiments (for example, 980 amino acids for thepeptide encoded by SEQ ID NO:1). Fragments of a nucleotide sequence thatare useful as hybridization probes or PCR primers generally need notencode a biologically active portion of a protein.

As used herein, “full-length sequence,” in reference to a specifiedpolynucleotide, means having the entire nucleic acid sequence of anative sequence. “Native sequence” is intended to mean an endogenoussequence, i.e., a non-engineered sequence found in an organism's genome.

Thus, a fragment of a nucleotide sequence of the embodiments may encodea biologically active portion of a polypeptide, or it may be a fragmentthat can be used as a hybridization probe or PCR primer using methodsdisclosed below. A biologically active portion of an antipathogenicpolypeptide can be prepared by isolating a portion of one of thenucleotide sequences of the embodiments, expressing the encoded portionof the protein and assessing the ability of the encoded portion of theprotein to confer or enhance fungal resistance in a plant. Nucleic acidmolecules that are fragments of a nucleotide sequence of the embodimentscomprise at least about 15, about 20, about 50, about 75, about 100, orabout 150 nucleotides, or up to the number of nucleotides present in afull-length nucleotide sequence disclosed herein (for example, 4212nucleotides for SEQ ID NO: 1).

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more internal sites within the nativepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the native polynucleotide. As used herein, a “native”polynucleotide or polypeptide comprises a naturally occurring nucleotidesequence or amino acid sequence, respectively. One of skill in the artwill recognize that variants of the nucleic acids of the embodimentswill be constructed such that the open reading frame is maintained. Forpolynucleotides, conservative variants include those sequences that,because of the degeneracy of the genetic code, encode the amino acidsequence of one of the polypeptides of the embodiments. Naturallyoccurring allelic variants such as these can be identified with the useof well-known molecular biology techniques, as, for example, withpolymerase chain reaction (PCR) and hybridization techniques as outlinedbelow. Variant polynucleotides also include synthetically derivedpolynucleotides, such as those generated, for example, by usingsite-directed mutagenesis but which still encode a protein of theembodiments. Generally, variants of a particular polynucleotide of theembodiments will have at least about 40%, about 45%, about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99% or more sequence identity to thatparticular polynucleotide as determined by sequence alignment programsand parameters described elsewhere herein.

Variants of a particular polynucleotide of the embodiments (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Thus, for example, isolated polynucleotides that encodea polypeptide with a given percent sequence identity to the polypeptideof SEQ ID NOs: 3 or 246 are disclosed. Percent sequence identity betweenany two polypeptides can be calculated using sequence alignment programsand parameters described elsewhere herein. Where any given pair ofpolynucleotides of the embodiments is evaluated by comparison of thepercent sequence identity shared by the two polypeptides they encode,the percent sequence identity between the two encoded polypeptides is atleast about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99% or more sequence identity.

“Variant” protein is intended to mean a protein derived from the nativeprotein by deletion or addition of one or more amino acids at one ormore internal sites in the native protein and/or substitution of one ormore amino acids at one or more sites in the native protein. Variantproteins encompassed by the embodiments are biologically active, that isthey continue to possess the desired biological activity of the nativeprotein, that is, the ability to confer or enhance plant fungal pathogenresistance as described herein. Such variants may result, for example,from genetic polymorphism or from human manipulation. Biologicallyactive variants of a native protein of the embodiments will have atleast about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99% or more sequence identity to the amino acid sequence for thenative protein as determined by sequence alignment programs andparameters described elsewhere herein. A biologically active variant ofa protein of the embodiments may differ from that protein by as few asabout 1-15 amino acid residues, as few as about 1-10, such as about6-10, as few as about 5, as few as 4, 3, 2, or even 1 amino acidresidue.

The proteins of the embodiments may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants and fragments of theantipathogenic proteins can be prepared by mutations in the DNA. Methodsfor mutagenesis and polynucleotide alterations are well known in theart. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S.Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques inMolecular Biology (MacMillan Publishing Company, New York) and thereferences cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al. (1978) Atlas ofProtein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.), herein incorporated by reference. Conservative substitutions,such as exchanging one amino acid with another having similarproperties, may be optimal.

Thus, the genes and polynucleotides of the embodiments include bothnaturally occurring sequences as well as mutant forms. Likewise, theproteins of the embodiments encompass both naturally occurring proteinsas well as variations and modified forms thereof. Such variants willcontinue to possess the desired ability to confer or enhance plantfungal pathogen resistance. Obviously, the mutations that will be madein the DNA encoding the variant must not place the sequence out ofreading frame and optimally will not create complementary regions thatcould produce secondary mRNA structure. See, EP Patent No. 0075444.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by screening transgenic plants which have been transformedwith the variant protein to ascertain the effect on the ability of theplant to resist fungal pathogenic attack.

Variant polynucleotides and proteins also encompass sequences andproteins derived from mutagenic or recombinogenic procedures, includingand not limited to procedures such as DNA shuffling. One of skill in theart could envision modifications that would alter the range of pathogensto which the protein responds. With such a procedure, one or moredifferent protein coding sequences can be manipulated to create a newprotein possessing the desired properties. In this manner, libraries ofrecombinant polynucleotides are generated from a population of relatedsequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between the protein geneof the embodiments and other known protein genes to obtain a new genecoding for a protein with an improved property of interest, such asincreased ability to confer or enhance plant fungal pathogen resistance.Strategies for such DNA shuffling are known in the art. See, forexample, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech.15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al.(1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998)Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

The polynucleotides of the embodiments can be used to isolatecorresponding sequences from other organisms, particularly other plants.In this manner, methods such as PCR, hybridization, and the like can beused to identify such sequences based on their sequence homology to thesequences set forth herein. Sequences isolated based on their sequenceidentity to the entire sequences set forth herein or to variants andfragments thereof are encompassed by the embodiments. Such sequencesinclude sequences that are orthologs of the disclosed sequences.“Orthologs” is intended to mean genes derived from a common ancestralgene and which are found in different species as a result of speciation.Genes found in different species are considered orthologs when theirnucleotide sequences and/or their encoded protein sequences share atleast about 60%, about 70%, about 75%, about 80%, about 85%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, or greater sequence identity. Functions oforthologs are often highly conserved among species. Thus, isolatedpolynucleotides that encode for a protein that confers or enhancesfungal plant pathogen resistance and that hybridize under stringentconditions to the sequences disclosed herein, or to variants orfragments thereof, are encompassed by the embodiments.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, and are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

In hybridization techniques, all or part of a known polynucleotide isused as a probe that selectively hybridizes to other correspondingpolynucleotides present in a population of cloned genomic DNA fragmentsor cDNA fragments (i.e., genomic or cDNA libraries) from a chosenorganism. The hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments, or other oligonucleotides, and may be labeledwith a detectable group such as ³²P, or any other detectable marker.Thus, for example, probes for hybridization can be made by labelingsynthetic oligonucleotides based on the polynucleotides of theembodiments. Methods for preparation of probes for hybridization and forconstruction of cDNA and genomic libraries are generally known in theart and are disclosed in Sambrook et al. (1989) supra.

For example, an entire polynucleotide disclosed herein, or one or moreportions thereof, may be used as a probe capable of specificallyhybridizing to corresponding polynucleotides and messenger RNAs. Toachieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique and are optimally at leastabout 10 nucleotides in length, at least about 15 nucleotides in length,or at least about 20 nucleotides in length. Such probes may be used toamplify corresponding polynucleotides from a chosen organism by PCR.This technique may be used to isolate additional coding sequences from adesired organism or as a diagnostic assay to determine the presence ofcoding sequences in an organism. Hybridization techniques includehybridization screening of plated DNA libraries (either plaques orcolonies; see, for example, Sambrook et al. (1989) supra.

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,optimally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a final wash in 0.1×SSC at 60 to 65° C. for at least 30minutes. Optionally, wash buffers may comprise about 0.1% to about 1%SDS. Duration of hybridization is generally less than about 24 hours,usually about 4 to about 12 hours. The duration of the wash time will beat least a length of time sufficient to reach equilibrium.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the thermal melting point (T_(m))can be approximated from the equation of Meinkoth and Wahl (1984) Anal.Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. T_(m) is reduced byabout 1° C. for each 1% of mismatching; thus, T_(m), hybridization,and/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with ≧90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the T_(m) for thespecific sequence and its complement at a defined ionic strength and pH.However, severely stringent conditions can utilize a hybridizationand/or wash at 1, 2, 3, or 4° C. lower than the T_(m); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the T_(m); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe T_(m). Using the equation, hybridization and wash compositions, anddesired T_(m), those of ordinary skill will understand that variationsin the stringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), itis optimal to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds.(1995) Current Protocols in Molecular Biology, Chapter 2 (GreenePublishing and Wiley-Interscience, New York). See Sambrook et al. (1989)supra.

Various procedures can be used to check for the presence or absence of aparticular sequence of DNA, RNA, or a protein. These include, forexample, Southern blots, northern blots, western blots, and ELISAanalysis. Techniques such as these are well known to those of skill inthe art and many references exist which provide detailed protocols. Suchreferences include Sambrook et al. (1989) supra, and Crowther, J. R.(2001), The ELISA Guidebook, Humana Press, Totowa, N.J., USA.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides or polypeptides: (a) “referencesequence,” (b) “comparison window,” (c) “sequence identity,” and, (d)“percentage of sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twopolynucleotides. Generally, the comparison window is at least about 20contiguous nucleotides in length, and optionally can be about 30, about40, about 50, about 100, or longer. Those of skill in the art understandthat to avoid a high similarity to a reference sequence due to inclusionof gaps in the polynucleotide sequence a gap penalty is typicallyintroduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, and are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of theembodiments. BLAST protein searches can be performed with the BLASTXprogram, score=50, wordlength=3, to obtain amino acid sequenceshomologous to a protein or polypeptide of the embodiments. To obtaingapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0)can be utilized as described in Altschul et al. (1997) Nucleic AcidsRes. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used toperform an iterated search that detects distant relationships betweenmolecules. See Altschul et al. (1997) supra. When utilizing BLAST,Gapped BLAST, PSI-BLAST, the default parameters of the respectiveprograms (e.g., BLASTN for nucleotide sequences, BLASTX for proteins)can be used. See the website at ncbi.nlm.nih.gov. Alignment may also beperformed manually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using Gap Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using Gap Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the GCG Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, and no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo polynucleotides or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The use of the term “polynucleotide” is not intended to limit theembodiments to polynucleotides comprising DNA. Those of ordinary skillin the art will recognize that polynucleotides can compriseribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the embodiments also encompass all forms of sequencesincluding, and not limited to, single-stranded forms, double-strandedforms, and the like.

Isolated polynucleotides of the embodiments can be incorporated intorecombinant DNA constructs capable of introduction into and replicationin a host cell. A “vector” may be such a construct that includes areplication system and sequences that are capable of transcription andtranslation of a polypeptide-encoding sequence in a given host cell. Anumber of vectors suitable for stable transfection of plant cells or forthe establishment of transgenic plants have been described in, e.g.,Pouwels et al, Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Flevin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

The terms “recombinant construct,” “expression cassette,” “expressionconstruct,” “chimeric construct,” “construct,” “recombinant DNAconstruct” and “recombinant DNA fragment” are used interchangeablyherein and are nucleic acid fragments. A recombinant construct comprisesan artificial combination of nucleic acid fragments, including, and notlimited to, regulatory and coding sequences that are not found togetherin nature. For example, a recombinant DNA construct may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source and arranged in a manner different than that foundin nature. Such construct may be used by itself or may be used inconjunction with a vector. If a vector is used then the choice of vectoris dependent upon the method that will be used to transform host cellsas is well known to those skilled in the art. For example, a plasmidvector can be used. The skilled artisan is well aware of the geneticelements that must be present on the vector in order to successfullytransform, select and propagate host cells comprising any of theisolated nucleic acid fragments of the embodiments. Screening to obtainlines displaying the desired expression level and pattern of thepolynucleotides or of the Rcg1 locus may be accomplished byamplification, Southern analysis of DNA, northern analysis of mRNAexpression, immunoblotting analysis of protein expression, phenotypicanalysis, and the like.

The term “recombinant DNA construct” refers to a DNA construct assembledfrom nucleic acid fragments obtained from different sources. The typesand origins of the nucleic acid fragments may be very diverse.

In some embodiments, expression cassettes comprising a promoter operablylinked to a heterologous nucleotide sequence of the embodiments arefurther provided. The expression cassettes of the embodiments find usein generating transformed plants, plant cells, and microorganisms and inpracticing the methods for inducing plant fungal pathogen resistancedisclosed herein. The expression cassette will include 5′ and 3′regulatory sequences operably linked to a polynucleotide of theembodiments. “Operably linked” is intended to mean a functional linkagebetween two or more elements. “Regulatory sequences” refer tonucleotides located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and which mayinfluence the transcription, RNA processing, stability, or translationof the associated coding sequence. Regulatory sequences may include, andare not limited to, promoters, translation leader sequences, introns,and polyadenylation recognition sequences. For example, an operablelinkage between a polynucleotide of interest and a regulatory sequence(a promoter, for example) is functional link that allows for expressionof the polynucleotide of interest. Operably linked elements may becontiguous or non-contiguous. When used to refer to the joining of twoprotein coding regions, by operably linked is intended that the codingregions are in the same reading frame. The cassette may additionallycontain at least one additional gene to be cotransformed into theorganism. Alternatively, the additional gene(s) can be provided onmultiple expression cassettes. Such an expression cassette is providedwith a plurality of restriction sites and/or recombination sites forinsertion of the polynucleotide that encodes an antipathogenicpolypeptide to be under the transcriptional regulation of the regulatoryregions. The expression cassette may additionally contain selectablemarker genes.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional initiation region (i.e., a promoter),translational initiation region, a polynucleotide of the embodiments, atranslational termination region and, optionally, a transcriptionaltermination region functional in the host organism. The regulatoryregions (i.e., promoters, transcriptional regulatory regions, andtranslational termination regions) and/or the polynucleotide of theembodiments may be native/analogous to the host cell or to each other.Alternatively, the regulatory regions and/or the polynucleotide of theembodiments may be heterologous to the host cell or to each other. Asused herein, “heterologous” in reference to a sequence is a sequencethat originates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention. For example, a promoteroperably linked to a heterologous polynucleotide is from a speciesdifferent from the species from which the polynucleotide was derived,or, if from the same/analogous species, one or both are substantiallymodified from their original form and/or genomic locus, or the promoteris not the native promoter for the operably linked polynucleotide.

The optionally included termination region may be native with thetranscriptional initiation region, may be native with the operablylinked polynucleotide of interest, may be native with the plant host, ormay be derived from another source (i.e., foreign or heterologous) tothe promoter, the polynucleotide of interest, the host, or anycombination thereof. Convenient termination regions are available fromthe Ti-plasmid of A. tumefaciens, such as the octopine synthase andnopaline synthase termination regions. See also Guerineau et al. (1991)Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfaconet al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989)Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic AcidsRes. 15:9627-9639. In particular embodiments, the potato proteaseinhibitor II gene (PinII) terminator is used. See, for example, Keil etal. (1986) Nucl. Acids Res. 14:5641-5650; and An et al. (1989) PlantCell 1:115-122, herein incorporated by reference in their entirety.

A number of promoters can be used in the practice of the embodiments,including the native promoters of the polynucleotide sequences ofinterest. The promoters can be selected based on the desired outcome. Awide range of plant promoters are discussed in the recent review ofPotenza et al. (2004) In Vitro Cell Dev Biol-Plant 40:1-22, hereinincorporated by reference. For example, the nucleic acids can becombined with constitutive, tissue-preferred, pathogen-inducible, orother promoters for expression in plants. Such constitutive promotersinclude, for example, the core promoter of the Rsyn7 promoter and otherconstitutive promoters disclosed in WO 99/43838 and U.S. Pat. No.6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171);ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 andChristensen et al. (1992) Plant Mol. Biol. 18:675-689); PEMU (Last etal. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984)EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and thelike. Other constitutive promoters include, for example, U.S. Pat. Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463; 5,608,142; and 6,177,611.

It may sometimes be beneficial to express the genes from an induciblepromoter, particularly from a pathogen-inducible promoter. Suchpromoters include those from pathogenesis-related proteins (PRproteins), which are induced following infection by a pathogen; e.g., PRproteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, forexample, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Ukneset al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol.Virol. 4:111-116. See also WO 99/43819, herein incorporated byreference.

Of interest are promoters that result in expression of a protein locallyat or near the site of pathogen infection. See, for example, Marineau etal. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989) MolecularPlant-Microbe Interactions 2:325-331; Somsisch et al. (1986) Proc. Natl.Acad. Sci. USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet.2:93-98; and Yang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. Seealso, Chen et al. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc.Natl. Acad. Sci. USA 91:2507-2511; Warner et al. (1993) Plant J.3:191-201; Siebertz et al. (1989) Plant Cell 1:961-968; U.S. Pat. No.5,750,386 (nematode-inducible); and the references cited therein. Ofparticular interest is the inducible promoter for the maize PRms gene,whose expression is induced by the pathogen Fusarium moniliforme (see,for example, Cordero et al. (1992) Physiol. Mol. Plant. Path.41:189-200).

Additionally, as pathogens find entry into plants through wounds orinsect damage, a wound-inducible promoter may be used in theconstructions of the embodiments. Such wound-inducible promoters includepotato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev.Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2(Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurlet al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) PlantMol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76);MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like,herein incorporated by reference.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, and are not limited to, the maize ln2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced expressionof the polypeptides of the embodiments within a particular plant tissue.For example, a tissue-preferred promoter may be used to express apolypeptide in a plant tissue where disease resistance is particularlyimportant, such as, for example, the roots, the stalk or the leaves.Tissue-preferred promoters include Yamamoto et al. (1997) Plant J.12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803;Hansen et al. (1997) Mol. Gen. Genet. 254(3):337-343; Russell et al.(1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) PlantPhysiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol.112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524;Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994)Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol.Biol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J.4(3):495-505. Such promoters can be modified, if necessary, for weakexpression.

Vascular tissue-preferred promoters are known in the art and includethose promoters that selectively drive protein expression in, forexample, xylem and phloem tissue. Vascular tissue-preferred promotersinclude, and are not limited to, the Prunus serotina prunasin hydrolasegene promoter (see, e.g., International Publication No. WO 03/006651),and also those found in U.S. patent application Ser. No. 10/109,488.

Stalk-preferred promoters may be used to drive expression of apolypeptide of the embodiments. Exemplary stalk-preferred promotersinclude the maize MS8-15 gene promoter (see, for example, U.S. Pat. No.5,986,174 and International Publication No. WO 98/00533), and thosefound in Graham et al. (1997) Plant Mol Biol 33(4): 729-735.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) PlantPhysiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al.(1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root-preferred promoters are known and can be selected from the manyavailable from the literature or isolated de novo from variouscompatible species. See, for example, Hire et al. (1992) Plant Mol.Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene);Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger et al.(1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao etal. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encodingcytosolic glutamine synthetase (GS), which is expressed in roots androot nodules of soybean). See also Bogusz et al. (1990) Plant Cell2(7):633-641, where two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa are described. Thepromoters of these genes were linked to a β-glucuronidase reporter geneand introduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach and Aoyagi (1991) describe their analysisof the promoters of the highly expressed rolC and rolD root-inducinggenes of Agrobacterium rhizogenes (see Plant Science (Limerick)79(1):69-76). They concluded that enhancer and tissue-preferred DNAdeterminants are dissociated in those promoters. Teeri et al. (1989)used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, an especially desirablecombination of characteristics for use with an insecticidal orlarvicidal gene (see EMBO J. 8(2):343-350). The TR1′ gene, fused tonptII (neomycin phosphotransferase II) showed similar characteristics.Additional root-preferred promoters include the VfENOD-GRP3 genepromoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and rolBpromoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691. See alsoU.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836;5,110,732; and 5,023,179.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, and are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphatesynthase) (see WO 00/11177 and U.S. Pat. No. 6,225,529; hereinincorporated by reference). Gamma-zein is a preferred endosperm-specificpromoter. Glob-1 is a preferred embryo-specific promoter. For dicots,seed-specific promoters include, and are not limited to, beanβ-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and thelike. For monocots, seed-specific promoters include, and are not limitedto, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken1, shrunken 2, globulin 1, etc. See also WO 00/12733, whereseed-preferred promoters from end1 and end2 genes are disclosed; hereinincorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

Expression cassettes may additionally contain 5′ leader sequences. Suchleader sequences can act to enhance translation. Translation leaders areknown in the art and include: picornavirus leaders, for example, EMCVleader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al.(1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, forexample, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus), and humanimmunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991)Nature 353:90-94); untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) inMolecular Biology of RNA, ed. Cech (Lissi, N.Y.), pp. 237-256); andmaize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991)Virology 81:382-385). See also, Della-Cioppa et al. (1987) PlantPhysiol. 84:965-968. Other methods known to enhance translation can alsobe utilized, for example, introns, and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su et al. (2004)Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. CellScience 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), andyellow florescent protein (PhiYFP™ from Evrogen, see, Bolte et al.(2004) J. Cell Science 117:943-54). For additional selectable markers,see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511;Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318;Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol.6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.(1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge etal. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad.Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl.Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidtet al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724. Such disclosures are herein incorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the embodiments.

The genes of the embodiments can be expressed as a transgene in order tomake plants resistant to Cg. Using the different promoters describedelsewhere in this disclosure, this will allow their expression in amodulated form in different circumstances. For example, one might desirehigher levels of expression in stalks to enhance resistance to Cg-causedstalk rot. In environments where Cg-caused leaf blight is more of aproblem, lines with higher expression levels in leaves could be used.However, one can also insert the entire genes, both native promoter andcoding sequence, as a transgene. Finally, using the genes of theembodiments as a transgenes will allow quick combination with othertraits, such as insect or herbicide resistance.

In certain embodiments the nucleic acid sequences of the embodiments canbe stacked with any combination of polynucleotide sequences of interestin order to create plants with a desired phenotype. This stacking may beaccomplished by a combination of genes within the DNA construct, or bycrossing a line containing Rcg1 and Rcg1b (as transgenes or as anintrogressed locus), with another line that comprises the combination.For example, the polynucleotides of the embodiments may be stacked withany other polynucleotides of the embodiments, or with other genes. Thecombinations generated can also include multiple copies of any one ofthe polynucleotides of interest. The polynucleotides of the embodimentscan also be stacked with any other gene or combination of genes toproduce plants with a variety of desired trait combinations includingand not limited to traits desirable for animal feed such as high oilgenes (e.g., U.S. Pat. No. 6,232,529); balanced amino acids (e.g.hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and5,703,409); barley high lysine (Williamson et al. (1987) Eur. J.Biochem. 165:99-106; and WO 98/20122); and high methionine proteins(Pedersen et al. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988)Gene 71:359; and Musumura et al. (1989) Plant Mol. Biol. 12: 123));increased digestibility (e.g., modified storage proteins (U.S.application Ser. No. 10/053,410, filed Nov. 7, 2001); and thioredoxins(U.S. application Ser. No. 10/005,429, filed Dec. 3, 2001)), thedisclosures of which are herein incorporated by reference. Thepolynucleotides of the embodiments can also be stacked with traitsdesirable for insect, disease or herbicide resistance (e.g., Bacillusthuringiensis toxic proteins (U.S. Pat. Nos. 5,366,892; 5,747,450;5,737,514; 5,723,756; 5,593,881; Geiser et al (1986) Gene 48:109);lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825); fumonisindetoxification genes (U.S. Pat. No. 5,792,931); avirulence and diseaseresistance genes (Jones et al. (1994) Science 266:789; Martin et al.(1993) Science 262:1432; Mindrinos et al. (1994) Cell 78:1089);acetolactate synthase (ALS) mutants that lead to herbicide resistancesuch as the S4 and/or Hra mutations; inhibitors of glutamine synthasesuch as phosphinothricin or basta (e.g., bar gene); and glyphosateresistance (EPSPS genes, GAT genes such as those disclosed in U.S.Patent Application Publication US2004/0082770, also WO02/36782 andWO03/092360)); and traits desirable for processing or process productssuch as high oil (e.g., U.S. Pat. No. 6,232,529); modified oils (e.g.,fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516));modified starches (e.g., ADPG pyrophosphorylases (AGPase), starchsynthases (SS), starch branching enzymes (SBE) and starch debranchingenzymes (SDBE)); and polymers or bioplastics (e.g., U.S. Pat. No.5,602,321; beta-ketothiolase, polyhydroxybutyrate synthase, andacetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol.170:5837-5847) facilitate expression of polyhydroxyalkanoates (PHAs)),the disclosures of which are herein incorporated by reference. One couldalso combine the polynucleotides of the embodiments with polynucleotidesproviding agronomic traits such as male sterility (e.g., see U.S. Pat.No. 5,583,210), stalk strength, flowering time, or transformationtechnology traits such as cell cycle regulation or gene targeting (e.g.WO 99/61619; WO 00/17364; WO 99/25821), the disclosures of which areherein incorporated by reference.

These stacked combinations can be created by any method including andnot limited to cross breeding plants by any conventional or TOPCROSS®methodology, or genetic transformation. If the traits are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant.

As shown in the Examples, the Rcg1 locus contains two components, Rcg1and Rcg1b, which when expressed together provide the phenotype of thepresent invention. The skilled artisan will recognize that if the genesare to be used transgenically, this could be done on a single chimericconstruct inserted into a plant cell to make a transgenic plant, but itcould also be done by inserting the two genes as separate chimericconstructs into separate plant cells to make two transgenic plant lines.These could then be crossed and progeny lines carrying both genesselected using the methods described herein to obtain lines resistant tothe pathogen. If one were to find a line in nature which expressed onlyone or the other gene, one could use a chimeric construct containingonly the missing gene to make a transgenic line expressing both genes.

The methods of the embodiments may involve, and are not limited to,introducing a polypeptide or polynucleotide into a plant. “Introducing”is intended to mean presenting to the plant the polynucleotide. In someembodiments, the polynucleotide will be presented in such a manner thatthe sequence gains access to the interior of a cell of the plant,including its potential insertion into the genome of a plant. Themethods of the embodiments do not depend on a particular method forintroducing a sequence into a plant, only that the polynucleotide gainsaccess to the interior of at least one cell of the plant. Methods forintroducing polynucleotides into plants are known in the art including,and not limited to, stable transformation methods, transienttransformation methods, and virus-mediated methods.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. “Host cell” refersthe cell into which transformation of the recombinant DNA constructtakes place and may include a yeast cell, a bacterial cell, and a plantcell. Examples of methods of plant transformation includeAgrobacterium-mediated transformation (De Blaere et al, 1987, Meth.Enzymol. 143:277) and particle-accelerated or “gene gun” transformationtechnology (Klein et al, 1987, Nature (London) 327:70-73; U.S. Pat. No.4,945,050), among others.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” or “transient expression” is intended to meanthat a polynucleotide is introduced into the plant and does notintegrate into the genome of the plant or a polypeptide is introducedinto a plant.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing polypeptides andpolynucleotides into plant cells include microinjection (Crossway et al.(1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986)Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediatedtransformation (U.S. Pat. Nos. 5,563,055- and 5,981,840), direct genetransfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballisticparticle acceleration (see, for example, Sanford et al., U.S. Pat. Nos.4,945,050; 5,879,918; 5,886,244; and 5,932,782; Tomes et al. (1995) inPlant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborgand Phillips (Springer-Verlag, Berlin); McCabe et al. (1988)Biotechnology 6:923-926); and Lec1 transformation (WO 00/28058). Alsosee, Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford etal. (1987) Particulate Science and Technology 5:27-37 (onion); Christouet al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl.Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat.Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) PlantPhysiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London)311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987)Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al.(1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman etal. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) PlantCell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet.84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992)Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant CellReports 12:250-255 and Christou and Ford (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens); all of which are hereinincorporated by reference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference. Briefly,the polynucleotide of the embodiments can be contained in transfercassette flanked by two non-identical recombination sites. The transfercassette is introduced into a plant have stably incorporated into itsgenome a target site which is flanked by two non-identical recombinationsites that correspond to the sites of the transfer cassette. Anappropriate recombinase is provided and the transfer cassette isintegrated at the target site. The polynucleotide of interest is therebyintegrated at a specific chromosomal position in the plant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting progeny having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the embodiments provides transformed seed (also referredto as “transgenic seed”) having a nucleotide construct of theembodiments, for example, an expression cassette of the embodiments,stably incorporated into their genome.

As used herein, the term “plant” can be a whole plant, any part thereof,or a cell or tissue culture derived from a plant. Thus, the term “plant”can refer to any of: whole plants, plant components or organs (includingbut not limited to embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, and the like), plant tissues, plant cells, plant protoplasts,plant cell tissue cultures from which maize plant can be regenerated,plant calli, plant clumps, and plant seeds. A plant cell is a cell of aplant, either taken directly from a seed or plant, or derived throughculture from a cell taken from a plant. Grain is intended to mean themature seed produced by commercial growers for purposes other thangrowing or reproducing the species. Progeny, variants, and mutants ofthe regenerated plants are also included within the scope of theembodiments, provided that these parts comprise the introducedpolynucleotides.

The embodiments of the invention may be used to confer or enhance fungalplant pathogen resistance or protect from fungal pathogen attack inplants, especially corn (Zea mays). It will protect different parts ofthe plant from attack by pathogens, including and not limited to stalks,ears, leaves, roots and tassels. Other plant species may also be ofinterest in practicing the embodiments of the invention, including, andnot limited to, other monocot crop plants.

Where appropriate, the polynucleotides may be optimized for increasedexpression in the transformed organism. For example, the polynucleotidescan be synthesized using plant-preferred codons for improved expression.See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference.

The embodiments of the present invention may be effective against avariety of plant pathogens, particularly fungal pathogens, such as, forexample, Colletotrichum, including Cg. The embodiments of the presentinvention may also be effective against maize stalk rot, includinganthracnose stalk rot, wherein the causative agent is Colletotrichum.Other plant pathogenic fungi and oomycetes (many of the latter of whichhave been historically been considered fungi although modern taxonomistshave now classified them separately) include, and are not limited to,the following: Soybeans: Phytophthora megasperma fsp. glycinea,Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum,Fusarium oxysporum, Diaporthe phaseolorum var. sojae (Phomopsis sojae),Diaporthe phaseolorum var. caulivora, Sclerotium rolfsii, Cercosporakikuchii, Cercospora sojina, Peronospora manshurica, Colletotrichumdematium (Colletotichum truncatum), Corynespora cassiicola, Septoriaglycines, Phyllosticta sojicola, Alternaria alternata, Microsphaeradiffusa, Fusarium semitectum, Phialophora gregata, Glomerella glycines,Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythiumdebaryanum, Fusarium solani; Canola: Albugo candida, Alternariabrassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotiniasclerotiorum, Mycosphaerella brassiccola, Pythium ultimum, Peronosporaparasitica, Fusarium roseum, Alternaria alternata; Alfalfa: Pythiumultimum, Pythium irregulare, Pythium splendens, Pythium debaryanum,Pythium aphanidermatum, Phytophthora megasperma, Peronosporatrifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis,Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium oxysporum,Verticillium albo-atrum, Aphanomyces euteiches, Stemphylium herbarum,Stemphylium alfalfae, Colletotrichum trifolii, Leptosphaerulinabriosiana, Uromyces striatus, Sclerotinia trifoliorum, Stagnosporameliloti, Stemphylium botryosum, Leptotrochila medicaginis; Wheat:Urocystis agropyri, Alternaria alternata, Cladosporium herbarum,Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochytatritici, Cephalosporium gramineum, Collotetrichum graminicola, Erysiphegraminis f.sp. tritici, Puccinia graminis f.sp. tritici, Pucciniarecondita f.sp. tritici, Puccinia striiformis, Pyrenophoratritici-repentis, Septoria nodorum, Septoria tritici, Septoria avenae,Pseudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctoniacerealis, Gaeumannomyces graminis var. tritici, Pythium aphanidermatum,Pythium arrhenomanes, Pythium ultimum, Bipolaris sorokiniana, Clavicepspurpurea, Tilletia tritici, Tilletia laevis, Ustilago tritici, Tilletiaindica, Rhizoctonia solani, Pythium arrhenomannes, Pythium gramicola,Pythium aphanidermatum; Sunflower: Plasmophora halstedii, Sclerotiniasclerotiorum, Septoria helianthi, Phomopsis helianthi, Alternariahelianthi, Alternaria zinniae, Botrytis cinerea, Phoma macdonaldii,Macrophomina phaseolina, Erysiphe cichoracearum, Rhizopus oryzae,Rhizopus arrhizus, Rhizopus stolonifer, Puccinia helianthi, Verticilliumdahliae, Erwinia carotovorum pv. carotovora, Cephalosporium acremonium,Phytophthora cryptogea, Albugo tragopogonis; Corn: Fusarium moniliformevar. subglutinans, Erwinia stewartii, Fusarium moniliforme,Stenocarpella maydi (Diplodia maydis), Pythium irregulare, Pythiumdebaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum,Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis O, T(Cochliobolus heterostrophus), Helminthosporium carbonum I, II & III(Cochliobolus carbonum), Exserohilum turcicum I, II & III,Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis,Kabatiella maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi,Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum,Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvulariainaequalis, Curvularia pallescens, Trichoderma viride, Claviceps sorghi,Erwinia chrysanthemi pv. zea, Erwinia carotovora, Diplodia macrospora,Sclerophthora macrospora, Peronosclerospora sorghi, Peronosclerosporaphilippinensis, Peronosclerospora maydis, Peronosclerospora sacchari,Sphacelotheca reiliana, Physopella zeae, Cephalosporium maydis,Cephalosporium acremonium; Sorghum: Exserohilum turcicum, Colletotrichumgraminicola (Glomerella graminicola), Cercospora sorghi, Gloeocercosporasorghi, Ascochyta sorghina, Puccinia purpurea, Macrophomina phaseolina,Perconia circinata, Fusarium moniliforme, Alternaria alternata,Bipolaris sorghicola, Helminthosporium sorghicola, Curvularia lunata,Phoma insidiosa, Ramulispora sorghi, Ramulispora sorghicola, Phyllacharasacchari, Sporisorium reilianum (Sphacelotheca reiliana), Sphacelothecacruenta, Sporisorium sorghi, Claviceps sorghi, Rhizoctonia solani,Acremonium strictum, Sclerophthona macrospora, Peronosclerospora sorghi,Peronosclerospora philippinensis, Sclerospora graminicola, Fusariumoxysporum, Pythium arrhenomanes, Pythium graminicola, etc.

“Germplasm” refers to genetic material of or from an individual (e.g., aplant), a group of individuals (e.g., a plant line, variety or family),or a clone derived from a line, variety, species, or culture. Thegermplasm can be part of an organism or cell, or can be separate fromthe organism or cell. In general, germplasm provides genetic materialwith a specific molecular makeup that provides a physical foundation forsome or all of the hereditary qualities of an organism or cell culture.As used herein, germplasm includes cells, seed or tissues from which newplants may be grown, or plant parts, such as leaves, stems, pollen, orcells, that can be cultured into a whole plant.

The term “allele” refers to one of two or more different nucleotidesequences that occur at a specific locus. A first allele is found on onechromosome, while a second allele occurs at the same position on thehomologue of that chromosome, e.g., as occurs for different chromosomesof a heterozygous individual, or between different homozygous orheterozygous individuals in a population. A “favorable allele” is theallele at a particular locus that confers, or contributes to, anagronomically desirable phenotype, e.g., resistance to Cg infection. Afavorable allele of a marker is a marker allele that segregates with thefavorable phenotype. A favorable allelic form of a chromosome segment isa chromosome segment that includes a nucleotide sequence thatcontributes to superior agronomic performance at one or more geneticloci physically located on the chromosome segment. “Allele frequency”refers to the frequency (proportion or percentage) of an allele within apopulation, or a population of lines. One can estimate the allelefrequency within a population by averaging the allele frequencies of asample of individuals from that population.

An allele “positively” correlates with a trait when it is linked to itand when presence of the allele is an indicator that the desired traitor trait form will occur in a plant comprising the allele. An allelenegatively correlates with a trait when it is linked to it and whenpresence of the allele is an indicator that a desired trait or traitform will not occur in a plant comprising the allele.

An individual is “homozygous” if the individual has only one type ofallele at a given locus (e.g., a diploid individual has a copy of thesame allele at a locus for each of two homologous chromosomes). Anindividual is “heterozygous” if more than one allele type is present ata given locus (e.g., a diploid individual with one copy each of twodifferent alleles). A special case of a heterozygous situation is whereone chromosome has an allele of a gene and the other chromosome lacksthat gene, locus or region completely—in other words, has a deletionrelative to the first chromosome. This situation is referred to as“hemizygous.” In this situation the allele on the chromosome lacking thegene, locus or region may also be referred to as the “null allele.” Theterm “homogeneity” indicates that members of a group have the samegenotype at one or more specific loci. In contrast, the term“heterogeneity” is used to indicate that individuals within the groupdiffer in genotype at one or more specific loci.

The embodiments provide not only a gene and its functional variants foruse in transgenic applications, but sequences and processes that allowthe Rcg1 and Rcg1b resistance genes to be moved between corn lines usingmarker assisted breeding. The embodiments also relate to plants producedby these processes that retain a truncated chromosomal intervalcomprising the Rcg1 and Rcg1b resistance genes.

A genetic map is a graphical representation of a genome (or a portion ofa genome such as a single chromosome) where the distances betweenlandmarks on a chromosome are measured by the recombination frequenciesbetween the landmarks. Recombinations between genetic landmarks can bedetected using a variety of molecular genetic markers (also calledmolecular markers) that are described in more detail herein.

For markers to be useful at detecting recombinations, they need todetect differences, or polymorphisms, within the population beingmonitored. For molecular markers, this means differences at the DNAlevel due to polynucleotide sequence differences (eg SSRs, RFLPs, FLPs,SNPs). The genomic variability can be of any origin, for example,insertions, deletions, duplications, repetitive elements, pointmutations, recombination events, or the presence and sequence oftransposable elements. Molecular markers can be derived from genomic orexpressed nucleic acids (e.g., ESTs). ESTs are generally well conservedwithin a species, while other regions of DNA (typically non-coding) tendto accumulate polymorphism, and therefore, can be more variable betweenindividuals of the same species. A large number of corn molecularmarkers are known in the art, and are published or available fromvarious sources, such as the Maize GDB internet resource and the ArizonaGenomics Institute internet resource run by the University of Arizona.

Molecular markers can be used in a variety of plant breedingapplications (eg see Staub et al. (1996) Hortscience 31: 729-741;Tanksley (1983) Plant Molecular Biology Reporter. 1: 3-8). One of themain areas of interest is to increase the efficiency of backcrossing andintrogressing genes using marker-assisted selection (MAS). A molecularmarker that demonstrates linkage with a locus affecting a desiredphenotypic trait provides a useful tool for the selection of the traitin a plant population. This is particularly true where the phenotype ishard to assay, e.g. many disease resistance traits, or, occurs at a latestage in the plants development, e.g. kernel characteristics. Since DNAmarker assays are less laborious, and take up less physical space, thanfield phenotyping, much larger populations can be assayed, increasingthe chances of finding a recombinant with the target segment from thedonor line moved to the recipient line. The closer the linkage, the moreuseful the marker, as recombination is less likely to occur between themarker and the gene causing the trait, which can result in falsepositives. Having flanking markers decreases the chances that falsepositive selection will occur as a double recombination event would beneeded. The ideal situation is to have a marker in the gene itself, sothat recombination can not occur between the marker and the gene. Such amarker is called a ‘perfect marker’.

When a gene is introgressed by MAS, it is not only the gene that isintroduced but also the flanking regions (Gepts. (2002). Crop Sci; 42:1780-1790). This is referred to as “linkage drag.” In the case where thedonor plant is highly unrelated to the recipient plant, as in the caseof the Rcg1 locus being introgressed from MP305, an exotic source, intoelite inbreds, these flanking regions carry additional genes that maycode for agronomically undesirable traits. This “linkage drag” may alsoresult in reduced yield or other negative agronomic characteristics evenafter multiple cycles of backcrossing into the elite corn line. This isalso sometimes referred to as “yield drag.” The size of the flankingregion can be decreased by additional backcrossing, although this is notalways successful, as breeders do not have control over the size of theregion or the recombination breakpoints (Young et al. (1998) Genetics120:579-585). In classical breeding it is usually only by chance thatrecombinations are selected that contribute to a reduction in the sizeof the donor segment (Tanksley et al. (1989). Biotechnology 7: 257-264).Even after 20 backcrosses in backcrosses of this type, one may expect tofind a sizeable piece of the donor chromosome still linked to the genebeing selected. With markers however, it is possible to select thoserare individuals that have experienced recombination near the gene ofinterest. In 150 backcross plants, there is a 95% chance that at leastone plant will have experienced a crossover within 1 cM of the gene,based on a single meiosis map distance. Markers will allow unequivocalidentification of those individuals. With one additional backcross of300 plants, there would be a 95% chance of a crossover within 1 cMsingle meiosis map distance of the other side of the gene, generating asegment around the target gene of less than 2 cM based on a singlemeiosis map distance. This can be accomplished in two generations withmarkers, while it would have required on average 100 generations withoutmarkers (See Tanksley et al., supra). When the exact location of a geneis known, a series of flanking markers surrounding the gene can beutilized to select for recombinations in different population sizes. Forexample, in smaller population sizes recombinations may be expectedfurther away from the gene, so more distal flanking markers would berequired to detect the recombination.

The availability of integrated linkage maps of the maize genomecontaining increasing densities of public maize markers has facilitatedmaize genetic mapping and MAS. See, e.g. the IBM2 Neighbors 4 map[online], [retrieved on Mar. 21, 2006]. Retrieved from the Maize GenomeDatabase website. See also: Sharopova et al 2002 Plant Mol Biol48:463-481; and Lee et al 2002 Plant Mol Biol 48: 453-461.

The key components to the implementation of MAS are: (i) Defining thepopulation within which the marker-trait association will be determined,which can be a segregating population, or a random or structuredpopulation; (ii) monitoring the segregation or association ofpolymorphic markers relative to the trait, and determining linkage orassociation using statistical methods; (iii) defining a set of desirablemarkers based on the results of the statistical analysis, and (iv) theuse and/or extrapolation of this information to the current set ofbreeding germplasm to enable marker-based selection decisions to bemade. The three types of markers described in this disclosure can beused in marker assisted selection protocols; simple sequence repeat(SSR, also known as microsatellite) markers, single nucleotidepolymorphism (SNP) markers and fragment length polymorphic (FLP)markers. SSRs can be defined as relatively short runs of tandemlyrepeated DNA with lengths of 6 bp or less (Tautz (1989) Nucleic AcidResearch 17: 6463-6471; Wang et al. (1994) Theoretical and AppliedGenetics, 88:1-6). Polymorphisms arise due to variation in the number ofrepeat units, probably caused by slippage during DNA replication(Levinson and Gutman (1987) Mol Biol Evol 4: 203-221). The variation inrepeat length may be detected by designing PCR primers to the conservednon-repetitive flanking regions (Weber and May (1989) Am J Hum Genet44:388-396). SSRs are highly suited to mapping and MAS as they aremulti-allelic, codominant, reproducible and amenable to high throughputautomation (Rafalski et al. (1996) Generating and using DNA markers inplants. In: Non-mammalian genomic analysis: a practical guide. Academicpress. pp 75-135).

For example, an SSR marker profile of MP305 is provided in Example 5herein. This marker profile was generated by gel electrophoresis of theamplification products generated by the primer pairs for these markers.Scoring of marker genotype is based on the size of the amplifiedfragment, which in this case was measured by the base pair weight of thefragment. While variation in the primer used or in laboratory procedurescan affect the reported base pair weight, relative values will remainconstant regardless of the specific primer or laboratory used. Thus,when comparing lines, the SSR profiles being compared should be obtainedfrom the same lab, so that the same primers and equipment is used. Forthis reason, when comparing plants or lines vis a vis specific markers,it is preferable to state that such plants or lines have the same (ordifferent) alleles at specified loci (e.g. one can say that if a plantdoes not comprise the MP305 derived chromosomal interval at or belowUMC15a, it will not comprise the same alleles as MP305 at all of theloci at or below UMC15a listed on Table 5b in Example 5). An SSR servicefor corn is available to the public on a contractual basis by DNALandmarks in Saint-Jean-sur-Richelieu, Quebec, Canada.

Various types of FLP markers can be generated. Most commonly,amplification primers are used to generate fragment lengthpolymorphisms. Such FLP markers are in many ways similar to SSR markers,except that the region amplified by the primers is not typically ahighly repetitive region. Still, the amplified region, or amplicon, willhave sufficient variability among germplasm, often due to insertions ordeletions, such that the fragments generated by the amplificationprimers can be distinguished among polymorphic individuals, and suchindels are known to occur frequently in maize (Bhattramakki et al.(2002). Plant Mol Biol 48, 539-547; Rafalski (2002b), supra). The term“indel” refers to an insertion or deletion, wherein one line may bereferred to as having an insertion relative to a second line, or thesecond line may be referred to as having a deletion relative to thefirst line. The MZA markers disclosed herein are examples of amplifiedFLP markers that have been selected because they are in close proximityto the Rcg1 and Rcg1b genes.

SNP markers detect single base pair nucleotide substitutions. Of all themolecular marker types, SNPs are the most abundant, thus having thepotential to provide the highest genetic map resolution (Bhattramakki etal. 2002 Plant Molecular Biology 48:539-547). SNPs can be assayed at aneven higher level of throughput than SSRs, in a so-called‘ultra-high-throughput’ fashion, as they do not require large amounts ofDNA and automation of the assay may be straight-forward. SNPs also havethe promise of being relatively low-cost systems. These three factorstogether make SNPs highly attractive for use in MAS. Several methods areavailable for SNP genotyping, including but not limited to,hybridization, primer extension, oligonucleotide ligation, nucleasecleavage, minisequencing and coded spheres. Such methods have beenreviewed in: Gut (2001) Hum Mutat 17 pp. 475-492; Shi (2001) Clin Chem47, pp. 164-172; Kwok (2000) Pharmacogenomics 1, pp. 95-100;Bhattramakki and Rafalski (2001) Discovery and application of singlenucleotide polymorphism markers in plants. In: R. J. Henry, Ed, PlantGenotyping: The DNA Fingerprinting of Plants, CABI Publishing,Wallingford. A wide range of commercially available technologies utilizethese and other methods to interrogate SNPs including MASSCODE™(Qiagen), INVADER™ (Third Wave Technologies, Madison, Wis.), SNAPSHOT®(Applied Biosystems), TAQMAN® (Applied Biosystems) and BEADARRAYS™(Illumina).

A number of SNPs together within a sequence, or across linked sequences,can be used to describe a haplotype for any particular genotype (Chinget al. (2002), BMC Genet. 3:19 pp Gupta et al. 2001, Rafalski (2002b),supra). Haplotypes can be more informative than single SNPs and can bemore descriptive of any particular genotype. For example, a single SNPmay be allele ‘T’ for MP305, but the allele ‘T’ might also occur in themaize breeding population being utilized for recurrent parents. In thiscase, a haplotype, e.g. a series of alleles at linked SNP markers, maybe more informative. Once a unique haplotype has been assigned to adonor chromosomal region, that haplotype can be used in that populationor any subset thereof to determine whether an individual has aparticular gene. See, for example, WO2003054229. Using automated highthroughput marker detection platforms known to those of ordinary skillin the art makes this process highly efficient and effective.

As described herein, many of the primers listed in Tables 1 and 2 canreadily be used as FLP markers to select for the Rcg1 locus. Theseprimers can also be used to convert these markers to SNP or otherstructurally similar or functionally equivalent markers (SSRs, CAPs,indels, etc), in the same regions. One very productive approach for SNPconversion is described by Rafalski (2002a) Current opinion in plantbiology 5 (2): 94-100 and also Rafalski (2002b) Plant Science 162:329-333. Using PCR, the primers are used to amplify DNA segments fromindividuals (preferably inbred) that represent the diversity in thepopulation of interest. The PCR products are sequenced directly in oneor both directions. The resulting sequences are aligned andpolymorphisms are identified. The polymorphisms are not limited tosingle nucleotide polymorphisms (SNPs), but also include indels, CAPS,SSRs, and VNTRs (variable number of tandem repeats). Specifically withrespect to the fine map information described herein, one can readilyuse the information provided herein to obtain additional polymorphicSNPs (and other markers) within the region amplified by the primerslisted in this disclosure. Markers within the described map region canbe hybridized to BACs or other genomic libraries, or electronicallyaligned with genome sequences, to find new sequences in the sameapproximate location as the described markers.

In addition to SSR's, FLPs and SNPs as described above, other types ofmolecular markers are also widely used, including but not limited toexpressed sequence tags (ESTs) and SSR markers derived from ESTsequences, and randomly amplified polymorphic DNA (RAPD). As usedherein, the term “Genetic Marker” shall refer to any type of nucleicacid based marker, including but not limited to, Restriction FragmentLength Polymorphism (RFLP), Simple Sequence Repeat (SSR), RandomAmplified Polymorphic DNA (RAPD), Cleaved Amplified PolymorphicSequences (CAPS) (Rafalski and Tingey, 1993, Trends in Genetics9:275-280), Amplified Fragment Length Polymorphism (AFLP) (Vos et al,1995, Nucleic Acids Res. 23:4407-4414), Single Nucleotide Polymorphism(SNP) (Brookes, 1999, Gene 234:177-186), Sequence CharacterizedAmplified Region (SCAR) (Paran and Michelmore, 1993, Theor. Appl. Genet.85:985-993), Sequence Tagged Site (STS) (Onozaki et al., 2004, Euphytica138:255-262), Single Stranded Conformation Polymorphism (SSCP) (Orita etal, 1989, Proc Natl Acad Sci USA 86:2766-2770), Inter-Simple SequenceRepeat (ISSR) (Blair et al, 1999, Theor. Appl. Genet. 98:780-792),Inter-Retrotransposon Amplified Polymorphism (IRAP),Retrotransposon-Microsatellite Amplified Polymorphism (REMAP) (Kalendaret al, 1999, Theor. Appl. Genet. 98:704-711), an RNA cleavage product(such as a Lynx tag) and the like.

More generically, the term “molecular marker” may be used to refer to agenetic marker, as defined above, or an encoded product thereof (e.g., aprotein) used as a point of reference when identifying a linked locus. Amarker can be derived from genomic nucleotide sequences or fromexpressed nucleotide sequences (e.g., from a spliced RNA, a cDNA, etc.),or from an encoded polypeptide. The term also refers to nucleic acidsequences complementary to or flanking the marker sequences, such asnucleic acids used as probes or primer pairs capable of amplifying themarker sequence. A “molecular marker probe” is a nucleic acid sequenceor molecule that can be used to identify the presence of a marker locus,e.g., a nucleic acid probe that is complementary to a marker locussequence. Alternatively, in some aspects, a marker probe refers to aprobe of any type that is able to distinguish (i.e., genotype) theparticular allele that is present at a marker locus. Nucleic acids are“complementary” when they specifically hybridize in solution, e.g.,according to Watson-Crick base pairing rules. Some of the markersdescribed herein are also referred to as hybridization markers whenlocated on an indel region, such as the non-colinear region describedherein. This is because the insertion region is, by definition, apolymorphism vis-à-vis a plant without the insertion. Thus, the markerneed only indicate whether the indel region is present or absent. Anysuitable marker detection technology may be used to identify such ahybridization marker, e.g. SNP technology is used in the examplesprovided herein.

A “genomic nucleic acid” is a nucleic acid that corresponds in sequenceto a heritable nucleic acid in a cell. Common examples include nucleargenomic DNA and amplicons thereof. A genomic nucleic acid is, in somecases, different from a spliced RNA, or a corresponding cDNA, in thatthe spliced RNA or cDNA is processed, e.g., by the splicing machinery,to remove introns. Genomic nucleic acids optionally comprisenon-transcribed (e.g., chromosome structural sequences, promoterregions, enhancer regions, etc.) and/or non-translated sequences (e.g.,introns), whereas spliced RNA/cDNA typically do not have non-transcribedsequences or introns. A “template nucleic acid” is a nucleic acid thatserves as a template in an amplification reaction (e.g., a polymerasebased amplification reaction such as PCR, a ligase mediatedamplification reaction such as LCR, a transcription reaction, or thelike). A template nucleic acid can be genomic in origin, oralternatively, can be derived from expressed sequences, e.g., a cDNA oran EST.

The term “amplifying” in the context of nucleic acid amplification isany process whereby additional copies of a selected nucleic acid (or atranscribed form thereof) are produced. Typical amplification methodsinclude various polymerase based replication methods, including thepolymerase chain reaction (PCR), ligase mediated methods such as theligase chain reaction (LCR) and RNA polymerase based amplification(e.g., by transcription) methods. An “amplicon” is an amplified nucleicacid, e.g., a nucleic acid that is produced by amplifying a templatenucleic acid by any available amplification method (e.g., PCR, LCR,transcription, or the like).

Isozyme profiles and linked morphological characteristics can, in somecases, also be indirectly used as markers. Even though they do notdirectly detect DNA differences, they are often influenced by specificgenetic differences. However, markers that detect DNA variation are farmore numerous and polymorphic than isozyme or morphological markers(Tanksley (1983) Plant Molecular Biology Reporter 1:3-8).

Sequence alignments or contigs may also be used to find sequencesupstream or downstream of the specific markers listed herein. These newsequences, close to the markers described herein, are then used todiscover and develop functionally equivalent markers.

For example, different physical and/or genetic maps are aligned tolocate equivalent markers not described within this disclosure but thatare within similar regions. These maps may be within the maize species,or even across other species that have been genetically or physicallyaligned with maize, such as rice, wheat, barley or sorghum.

As noted in Example 2, by using common sequences from the regionflanking the Rcg1 locus that hybridized to BACs in the Mo17 and the B73BAC libraries, the BACs from both libraries were lined up with BACs fromthe DE811ASR(BC5) homologous region flanking the Rcg1 locus in a tilingpath as shown in FIG. 9( a). The public B73 BACs, c0113f01 and c0117e18were identified as directly north and south, respectively, of the Rcg1locus.

With this information, an extended non-contiguous tiling path of B73BACs between genetic markers UMC2285 and UMC15a, UMC2285 and UMC2187,UMC1086 and UMC2200, or UMC2041 and UMC2200, can be created by aligninggenetic markers within this region with the physical map of the B73 BAC.Alignment information of the genetic and physical maps of B73 isobtained from the maize genome database of the Arizona GenomicsInstitute on the world wide web, accessed by entering the following webaddress prefixed by “www.”: genome.arizona.edu/fpc/maize/#webagcol. Inthe WebChrom view, one can select the genetic markers in the vicinity ofthe Rcg1 and Rcg1b genes and get a link to the physical contig wherethese genetic markers are located. By aligning the physical map in suchway with the genetic map one can find a plethora of B73 BACs in theregion between the chromosomal intervals defined by genetic markersUMC2285 and UMC15a, UMC2285 and UMC2187, UMC1086 and UMC2200, or UMC2041and UMC2200. The BACs can be used by one of ordinary skill in the art todevelop new markers for introgression of the Rcg1 locus into maizegermplasm. In particular, such genetic markers would be useful fortracking the Rcg1 locus in any lines into which the Rcg1 locus or Rcg1and Rcg1b genes have been introgressed, and for selecting for recurrentparent genome in a backcrossing program.

For example, in order to design polymorphic markers that will be usefulfor introgression and selection of the Rcg1 and Rcg1b genes, or the Rcg1locus in other maize germplasm, sequence information of the regionsurrounding the Rcg1 locus can be used. There are many B73 derivedbacterial artificial chromosomes (BACs) available in the region ofinterest from which sequence information can be obtained. An example ofBACs in the region of interest is shown in FIG. 21, which shows a contigon the B73 physical map that is homologous to the Rcg1 and Rcg1b regionin DE811ASR (BC5). Sequence information is obtained either throughinformation that is already publicly available (e.g. BAC end-sequence,sequence of Expressed Sequence Tags (ESTs) that hybridize to BACs inthis region, overgo probes that often relate to these ESTs, etc.) or byobtaining new sequence by directly sequencing BAC clones in this region.From this sequence one can determine which regions are most unique usingseveral different methods known to one of ordinary skill in the art. Forexample, by using gene prediction software or by comparing the sequenceusing BLAST® against all available maize sequence, one can select fornon-repetitive sequence. Low copy sequence can be used to develop a widearray of nucleic acid based markers. These markers are used to screenthe plant material in which the Rcg1 locus is present and the plantmaterial in which the Rcg1 locus is absent. If a marker outside of theRcg1 locus is desired, then the markers are used to screen the plantmaterial in which the Rcg1 locus is present and the plant material inwhich the Rcg1 locus is absent to determine if the marker is polymorphicin such germplasm. Polymorphic markers are then used for marker assistedintrogression and selection of the Rcg1 region and optimally alsorecurrent parent genome selection, in other maize germplasm. Thus, withthe location of the Rcg1 locus identified and its association withresistance to Colletotrichum established, one of ordinary skill in theart can utilize any number of existing markers, or readily develop newmarkers, that can be used introgress or identify the presence or absenceof the Rcg1 locus in germplasm, and to select for recurrent parentgenome in a backcrossing program.

On a genetic map, linkage of one molecular marker to a gene or anothermolecular marker is measured as a recombination frequency. In general,the closer two loci (e.g., two SSR markers) are on the genetic map, thecloser they lie to each other on the physical map. A relative geneticdistance (determined by crossing over frequencies, measured incentimorgans; cM) can be proportional to the physical distance (measuredin base pairs, e.g., kilobase pairs [kb] or mega-basepairs [Mbp]) thattwo linked loci are separated from each other on a chromosome. A lack ofprecise proportionality between cM and physical distance can result fromvariation in recombination frequencies for different chromosomalregions, e.g., some chromosomal regions are recombination “hot spots,”while others regions do not show any recombination, or only demonstraterare recombination events. Some of the introgression data and mappinginformation suggest that the region around the Rcg1 locus is one thatdoes have a high amount of recombination.

In general, the closer one marker is to another marker, whether measuredin terms of recombination or physical distance, the more strongly theyare linked. The closer a molecular marker is to a gene that encodes apolypeptide that imparts a particular phenotype (disease resistance),whether measured in terms of recombination or physical distance, thebetter that marker serves to tag the desired phenotypic trait. Ifpossible, the best marker is one within the gene itself, since it willalways remain linked with the gene causing the desired phenotype.

Genetic mapping variability can also be observed between differentpopulations of the same crop species, including maize. In spite of thisvariability in the genetic map that may occur between populations,genetic map and marker information derived from one population generallyremains useful across multiple populations in identification of plantswith desired traits, counter-selection of plants with undesirable traitsand in guiding MAS.

To locate equivalent markers across genetic maps, a mapping populationmay be used to confirm whether any such equivalent marker is within theregion described herein and therefore useful for selection of the Rcg1locus. Using this method, the equivalent marker, along with the markerslisted herein, are mapped on such mapping population. Any equivalentmarker that falls within the same region can be used to select for Rcg1.Mapping populations known in the art and that may be used for thispurpose include, but are not limited to, the IBM populations andT218×GT119 IF2 population described in Sharopova, N. et al. (2002) PlantMol Biol 48(5):463-481 and Lee, M. et al. (1999): Tools for highresolution genetic mapping in maize-status report. Proc. Plant AnimalGenome VII, January 17-21, 1999, San Diego, USA, P. 146; the UMC 98population, described in Davis, G. L. et al. (1999) Genetics152(3):1137-72 and in Davis, M. D. et al., (1998) The 1998 UMC MaizeGenetic Map: ESTs, Sequenced Core Markers, and Nonmaize Probes as aFoundation for Gene Discovery, Maize Genetics Conference Abstracts 40.

As used herein, “introgression” or “introgressing” shall refer to movinga gene or locus from one line to another by: (1) crossing individuals ofeach line to create a population; and (2) selecting individuals carryingthe desired gene or locus. Selection may be done phenotypically or usingmarkers (marker assisted selection). The individuals so selected areagain crossed (i.e., backcrossed) with the desired target line; theremay be two, three, four, five, six or more, or even ten or morebackcrosses. After each cross, the selection process is repeated. Forexample, the gene of the embodiments, or the locus containing it, may beintrogressed into a recurrent parent that is not resistant or onlypartially resistant, meaning that it is sensitive or susceptible orpartially so, to Cg. The recurrent parent line with the introgressedgene or locus then has enhanced or newly conferred resistance to Cg.This line into which the Rcg1 locus has been introgressed is referred toherein as an Rcg1 locus conversion.

The process of introgressing is often referred to as “backcrossing” whenthe process is repeated two or more times. In introgressing orbackcrossing, the “donor” parent refers to the parental plant with thedesired gene or locus to be introgressed. The “recipient” parent (usedone or more times) or “recurrent” parent (used two or more times) refersto the parental plant into which the gene or locus is beingintrogressed. For example, see Ragot, M. et al. (1995) Marker-assistedbackcrossing: a practical example, in Techniques et Utilisations desMarqueurs Moleculaires (Les Colloques, Vol. 72, pp. 45-56 and Openshawet al., (1994) Marker-assisted Selection in Backcross Breeding, Analysisof Molecular Marker Data, pp. 41-43. The initial cross gives rise to theF1 generation; the term “BC1” then refers to the second use of therecurrent parent, “BC2” refers to the third use of the recurrent parent,and so on.

In the case of the Rcg1 locus, where the sequences of the genesresponsible for its phenotypic effect and very nearby regions areavailable, DNA markers based on the genes themselves or closely linkedsequences can be developed for direct selection of the donor genes inthe recurrent parent background. While any polymorphic DNA sequence fromthe chromosomal region carrying the gene could be used, the sequencesprovided in the embodiments allow the use of DNA markers within or closeto the genes, minimizing false positive selection for the genes.Flanking markers limit the size of the donor genome fragments introducedinto the recipient background, thus minimizing so called “linkage drag,”meaning the introduction of undesirable sequences from the donor linethat could impact plant performance in otherwise elite germplasm. Theembodiments provide multiple examples of DNA markers that could be soused, and the person skilled in the art will be able to use the genomicsequences provided to create even more markers. An example is to usemarkers that hybridize (in the case of RFLP assays) or anneal (in thecase of PCR assays) specifically (exclusively) to sequences closelylinked, including within, the locus. In principle, sequences that alsohybridize or anneal elsewhere in the genome could be used if severalsuch markers are used in combination. When PCR reactions are used, inpractice the length of the primers used in the amplification reactionshould be at least about 15 nucleotides, but depending on the sequencesand hybridization conditions, any length that provides specificannealing can be used, such as about 16, about 17, about 18, about 19,about 20, about 21, about 22, about 23, about 24, about 25, about 26,about 27, about 28 or longer. For PCR reactions the term “anneal” iscommonly used, and as used herein it shall be understood to have thesame meaning as “hybridize.”

Thus, by using the markers and processes described herein, one mayproduce a plant comprising a truncated chromosomal interval comprisingthe Rcg1 locus and/or the Rcg1 and Rcg1b genes. The term “chromosomalinterval” or “chromosomal segment” refers to a contiguous linear span ofgenomic DNA that resides in planta on a single chromosome, usuallydefined with reference to two markers defining the end points of thechromosomal interval. The specified interval may include the markers atthe end points (e.g. one or more markers on or within the chromosomalinterval defined by marker A and marker B) or may exclude the markers atthe end points of the interval (e.g. one or more markers within thechromosomal interval defined by marker A and marker B). A truncatedchromosomal interval refers to a chromosomal interval that has beenreduced in size by selecting for one or more recombination events thathave reduced the size of the chromosomal interval. A “recombinationevent” refers to the occurrence of recombination between homologouschromosomes, and refers to a specific chromosomal location where such arecombination has occurred (e.g. a recombination of a chromosomalinterval internal to the end points of the chromosome will have arecombination event at each end of the chromosomal interval). Thetruncated chromosomal interval may be defined with reference to one orboth new markers at the end points of the segment. The length of twochromosomal segments may be measured by either centimorgans or basepairs. The genetic elements or genes located on a single chromosomalinterval are physically linked. The size of a chromosomal interval isnot particularly limited, but in the context of the embodiments of thepresent invention, generally the genetic elements located within asingle chromosomal interval are also genetically linked.

By using the processes of the embodiments, it is possible to select fora plant that comprises a truncated chromosomal interval comprising theRcg1 and Rcg1b genes. Specifically, with respect to the inventiondescribed in more detail in the examples below, the chromosomal intervalmay be reduced to a length of 12 cM or less, 10 cM or less, 8 cM orless, 6 cM or less, 4 cM or less, 3 cM or less, 2.5 cM or less, 2 cM orless, 1.5 cM or less, 1 cM or less, 0.75 cM or less, in each case asmeasured with respect to the map distances as shown on the IBM2Neighbors 4 genetic map as in effect on Mar. 21, 2006. As measured inbase pairs, the chromosomal interval may be reduced to a length of 15mbp or less, 10 mbp or less, 5 mbp or less, 3 mbp or less, 1 mpb orless, 500 kbp or less. One of ordinary skill in the art would understandthat it is undesirable to cause a break in the chromosomal region soproximal to the Rcg1 or Rcg1b coding sequences (e.g. within 5 kbp orless, within 4 kbp or less, 3 kbp or less, 2 kbp or less, 1 kbp orless), such that the promoter and other upstream regulatory elementswould be unlinked from the coding sequence.

The term “locus” generally refers to a genetically defined region of achromosome carrying a gene or, possibly, two or more genes so closelylinked that genetically they behave as a single locus responsible for aphenotype. When used herein with respect to Rcg1, the “Rcg1 locus” shallrefer to the defined region of the chromosome carrying the Rcg1 andRcg1b genes including their associated regulatory sequences, plus theregion surrounding the two genes that is non-colinear with B73, or anysmaller portion thereof that retains the Rcg1 and Rcg1b genes andassociated regulatory sequences. This locus has also been referred toelsewhere as the ASR locus, and will be referred to as the Rcg1 locushere.

A “gene” shall refer to a specific genetic coding region within a locus,including its associated regulatory sequences. The region encoding theRcg1 primary transcript, referred to herein as the “Rcg1 codingsequence”, will be used to define the position of the Rcg1 gene, and oneof ordinary skill in the art would understand that the associatedregulatory sequences will be within a distance of about 4 kb from theRcg1 coding sequence, with the promoter located upstream. The regionencoding the Rcg1b primary transcript, referred to herein as the “Rcg1bcoding sequence”, will be used to define the position of the Rcg1b gene,and one of ordinary skill in the art would understand that theassociated regulatory sequences will be within a distance of about 4 kb(i.e. about 4 kb, about 3 kb, about 2 kb, about 1 kb, or about 0.5 kb)from the Rcg1b coding sequence, with the promoter located upstream. Oneembodiment of the present invention is the isolation of the Rcg1 andRcg1b genes and the demonstration that they together are the genesresponsible for the phenotype conferred by the presence of the locus.

As will be appreciated by the skilled artisan, the structure of the Rcg1locus brings with it special considerations when using MAS to work withthis locus. It comprises two genes (Rcg1 and Rcg1b) located within lessthan a centimorgan of each other that are each necessary for thephenotype conferred by the presence of the locus. Physically the twogenes are approximately 200-300 kb apart. This means that the chances ofrecombination between the two genes, so that they are separated, is verylow, meaning less than 1%, on a chromosome the size of chromosome 4(approximately 250 megabases, or 250,000 kb). There are several reasonsfor this.

-   -   1. The physical distance represents about 0.1% (i.e., as a        fraction 0.001) of the length of the chromosome. If        recombination occurs randomly, the chance of a recombination        event in this region is clearly low.    -   2. While recombination frequencies are not identical at        different locations in the genome, the average physical distance        per centimorgan (1% recombination—see Suzuki, Griffiths, Miller        and Lewontin, (1986) An Introduction to Genetic Analysis, Third        Edition, Freeman & Company, New York, page 84) is approximately        1,000 kb. 200-300 kb would thus represent less than 1 cM, or        less than 1% recombination. The number of recombination events        found in Example 1 in the general region flanking the locus is        not unusually large.    -   3. As shown in Example 2, the locus is present on a chromosomal        region from MP305 that is not present in DE811 and in fact in        many other lines commonly used as breeding sources for        commercial lines. This region is referred to herein as the        noncolinear region. In such cases, there can be no recombination        because there is no homologous region on the other chromosome.        Consistent with this, in Example 1 recombination events were        found only outside of the region introgressed from MP305. This        specific reasoning will apply only to lines not containing a        segment homologous to the MP305 region, but the skilled artisan        will easily be able to determine this using the methods outlined        in Example 11.

Taken together, this means that while in principle, to avoid any chanceof separating the two genes, one should use markers outside the intervaldefined by the two genes, as a practical matter one could use markerswithin either gene, or in the interval between them, to follow andsuccessfully introgress both genes. The statistical chance of losing oneor the other gene is less than 1%, as described above, and even thatrisk can be avoided with the various assays for the presence of theindividual genes (including but not limited to gene specific PCRs,RT-PCR, westerns, ELISAs and other methods previously mentioned).Therefore, embodiments of this invention include both methods to use MASin a way that avoids any risk of losing either gene, by using markersflanking the interval defined by the genes, but also methods based onfollowing only one or the other gene, or the region between them,combined with the appropriate assays to make sure that both genes arepresent.

It should further be noted that because the two genes are so tightlylinked, previous workers would not have observed them as two genes. Thusthese two genes do not represent the two genetically separable genes onthe long arm of chromosome 4 that confer resistance to Cg previouslyreported (Toman, et al., (1993), Phytopathology, 83:981-986; Cowen, N etal. (1991) Maize Genetics Conference Abstracts 33). The two genestogether on the Rcg1 locus of the present invention may represent one ofthe two genetically separable loci, which have previously been reported,but which have not previously been associated with a particular gene orfinely mapped locus.

As used herein, “linked” or “linkage” (as distinguished from the term“operably linked”) shall refer to the genetic or physical linkage ofloci or genes. Loci or genes are considered genetically linked if therecombination frequency between them is less than about 50% asdetermined on a single meiosis map. They are progressively more linkedif the recombination frequency is about 40%, about 30%, about 20%, about10% or less, as determined on a single meiosis map. Two or more genesare physically linked (or syntenic) if they have been demonstrated to beon a single piece of DNA, such as a chromosome. Genetically linked geneswill in practice be physically linked (or syntenic), but the exactphysical distance (number of nucleotides) may not have been demonstratedyet. As used herein, the term “closely linked” refers to geneticallylinked markers within 15 cM or less, including without limitation 12 cMor less, 10 cM or less, 8 cM or less, 7 cM or less, 6 cM or less, 5 cMor less, 4 cM or less, 3 cM or less, 2 cM or less, 1 cM or less and 0.5cM or less as determined on the IBM2 neighbors 4 genetic map publiclyavailable on the Maize GDB website previously referenced in thisdisclosure. Distances 1 cM or less and 0.5 cM or less can also beconsidered very closely, or tightly, linked. A DNA sequence, such as ashort oligonucleotide representing a sequence within a locus or onecomplementary to it, is also linked to that locus.

A “line” or “strain” is a group of individuals of identical parentagethat are generally inbred to some degree and that are generallyhomozygous and homogeneous at most loci.

An “ancestral line” or “progenitor” is a parent line used as a source ofgenes, e.g., for the development of elite lines. “Progeny” are thedescendents of the ancestral line, and may be separated from theirancestors by many generations of breeding. For example, many elite linesare the progeny of B73 or Mo17. A “pedigree structure” defines therelationship between a descendant and each ancestor that gave rise tothat descendant. A pedigree structure can span one or more generations,describing relationships between the descendant and it's parents, grandparents, great-grand parents, etc.

An “elite line” or “elite variety” is an agronomically superior line orvariety that has resulted from many cycles of breeding and selection forsuperior agronomic performance. An “elite inbred line” is an elite linethat is an inbred, and that has been shown to be useful for producingsufficiently high yielding and agronomically fit hybrid varieties (an“elite hybrid variety”). Numerous elite lines and varieties areavailable and known to those of skill in the art of corn breeding.Similarly, “elite germplasm” is an agronomically superior germplasm,typically derived from and/or capable of giving rise to a plant withsuperior agronomic performance, such as an existing or newly developedelite line of corn.

In contrast, an “exotic corn line” or “exotic corn germplasm” isgermplasm derived from corn not belonging to an available elite line,elite variety or elite germplasm. In the context of a cross between twocorn plants, an exotic line or exotic germplasm is not closely relatedby descent to the elite line, elite variety or elite germplasm withwhich it is crossed. Most commonly, the exotic line or exotic germplasmis selected to introduce novel genetic elements (typically novelalleles) into a breeding program.

Units, prefixes, and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxyl orientation, respectively. Numeric ranges areinclusive of the numbers defining the range. Amino acids may be referredto herein by either their commonly known three letter symbols or by theone-letter symbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes. The above-defined terms are more fullydefined by reference to the specification as a whole.

With respect to map directions noted herein, instead of the terms 5′ and3′, the terms “north” and “above” are used (e.g., a marker north of theRcg1 locus refers to a marker above the Rcg1 locus, as determined withreference to the maps provided in a vertical orientation, such as FIGS.7 and 8, and to the left of the Rcg1 locus, as determined with referenceto maps provided in a horizontal orientation, such as FIG. 22).Likewise, the terms “south” and “below” are used (e.g. a marker south ofthe Rcg1 locus refers to a marker below the Rcg1 locus, as determinedwith reference to the vertically oriented maps provided herein, and tothe right of the Rcg1 locus, as determined with reference to thehorizontally oriented maps provided herein). More specifically, forexample, above the Rcg1 coding sequence refers to the chromosome above,or north of the primary transcript in SEQ ID NO: 1 (at about FLP110F),and below the Rcg1 coding sequence refers to the chromosome below orsouth of the primary transcript in SEQ ID NO: 1 (at about FLPA1R). SeeFIG. 26. The term “proximal” and “distal” are relative terms meaning,respectively, nearer and farther from a specified location (e.g., theRcg1b or Rcg1 genes) when used to compare two points on a map relativeto the specified location.

A phenotypic assay is an assay in which, instead of using molecularmarkers to follow a trait or characteristic, the phenotype of the plantwith respect to the trait or characteristic is checked. For any giventrait or characteristic the skilled artisan will be able to develop orfind in the literature numerous phenotypic assays. For example, twophenotypic assays for resistance to Colletotrichum graminicola are theleaf blight assays described in Example 12 and the stalk infection assaydescribed in Example 14. Other phenotypic assays for resistance toColletotrichum assays are possible.

The term “computer systems” refers generally to various automatedsystems used to perform some or all of the method steps describedherein. The term “instructions” refers to computer code that instructsthe computer system to perform some or all of the method steps. Inaddition to practicing some or all of the method steps, digital oranalog systems, e.g., comprising a digital or analog computer, can alsocontrol a variety of other functions such as a user viewable display(e.g., to permit viewing of method results by a user) and/or control ofoutput features (e.g., to assist in marker assisted selection or controlof automated field equipment).

Certain of the methods described herein are optionally (and typically)implemented via a computer program or programs (e.g., that store and canbe used to analyze molecular marker data). Thus, the embodiments providedigital systems, e.g., computers, computer readable media, and/orintegrated systems comprising instructions (e.g., embodied inappropriate software) for performing the methods herein. The digitalsystem will include information (data) corresponding to plant genotypesfor a set of genetic markers, and optionally, phenotypic values and/orfamily relationships. The system can also aid a user in performingmarker assisted selection for the Rcg1 locus according to the methodsherein, or can control field equipment which automates selection,harvesting, and/or breeding schemes.

Standard desktop applications such as word processing software (e.g.,MICROSOFT® WORD or COREL® WORDPERFECT®) and/or database software (e.g.,spreadsheet software such as MICROSOFT® EXCEL®, COREL® QUATTRO PRO®, ordatabase programs such as MICROSOFT® ACCESS™ or PARADOX®) can be adaptedto the embodiments by inputting data which is loaded into the memory ofa digital system, and performing an operation as noted herein on thedata. For example, systems can include the foregoing software having theappropriate genotypic data, and optionally pedigree data, used inconjunction with a user interface (e.g., a GUI in a standard operatingsystem such as a WINDOWS®, MACINTOSH® or LINUX® system) to perform anyanalysis noted herein, or simply to acquire data (e.g., in aspreadsheet) to be used in the methods herein. The computer can be,e.g., a PC (INTEL® x86 or PENTIUM® chip-compatible MS-DOS®, OS/2®,WINDOWS®, WINDOWS NT®, WINDOWS® 95, WINDOWS® 98, LINUX®,Apple®-compatible, MACINTOSH® compatible, POWER PC® compatible, or aUNIX® compatible (e.g., SUN™ work station) machine) or othercommercially common computer which is known to one of skill. Softwarefor performing association analysis and/or phenotypic value predictioncan be constructed by one of skill using a standard programming languagesuch as VISUAL BASIC®, FORTRAN®, Basic, JAVA®, or the like, according tothe methods herein.

Any system controller or computer optionally includes a monitor, whichcan include, e.g., a cathode ray tube (“CRT”) display, a flat paneldisplay (e.g., active matrix liquid crystal display, liquid crystaldisplay), or others. Computer circuitry is often placed in a box whichincludes numerous integrated circuit chips, such as a microprocessor,memory, interface circuits, and others. The box also optionally includesa hard disk drive, a floppy disk drive, a high capacity removable drivesuch as a writeable CD-ROM, and other common peripheral elements.Inputting devices such as a keyboard or mouse optionally provide forinput from a user and for user selection of genetic marker genotype,phenotypic value, or the like in the relevant computer system.

The computer typically includes appropriate software for receiving userinstructions, either in the form of user input into a set of parameterfields, e.g., in a GUI, or in the form of preprogrammed instructions,e.g., preprogrammed for a variety of different specific operations. Thesoftware then converts these instructions to an appropriate language forinstructing the system to carry out any desired operation. For example,a digital system can instruct selection of plants comprising certainmarkers, or control field machinery for harvesting, selecting, crossingor preserving crops according to the relevant method herein.

The invention can also be embodied within the circuitry of anapplication specific integrated circuit (ASIC) or programmable logicdevice (PLD). In such a case, the invention is embodied in a computerreadable descriptor language that can be used to create an ASIC or PLD.The invention can also be embodied within the circuitry or logicprocessors of a variety of other digital apparatus, such as PDAs, laptopcomputer systems, displays, image editing equipment, etc.

EXAMPLES

The embodiments of the invention are further defined in the followingexamples, in which all parts and percentages are by weight and degreesare Celsius, unless otherwise stated. It should be understood that theseexamples, while indicating embodiments of the invention, are given byway of illustration only. From the above discussion and these examples,one skilled in the art can ascertain the essential characteristics ofthe embodiments of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications to adaptit to various usages and conditions. Thus, various modifications of theembodiments of the invention in addition to those shown and describedherein will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. The disclosure of each reference set forthherein is incorporated by reference in its entirety. Examples 5 and 15are actual in part and prophetic in part. All other examples are actual.

Example 1 Fine Mapping of the Rcg1 Locus to a Specific Region of 4L

In order to map and clone the gene responsible for the resistance ofcorn line MP305 to Cg, lines had previously been created which differedas little as possible from each other genetically with the exception ofthe presence of the locus responsible for the resistant phenotype. Suchlines are called near isogenic lines. To this end, DE811 had beencrossed to MP305 and the progeny had been backcrossed to the sensitiveline DE811 three times, at each backcross selecting for resistance to Cgand otherwise for characteristics of DE811 (Weldekidan and Hawk, (1993),Maydica, 38:189-192). The resulting line was designated DE811ASR (BC3)(Weldekidan and Hawk, (1993) supra). This line was used as the startingpoint for the fine mapping of the Rcg1 locus. It was first necessary toknow roughly where in the maize genome it was located. Using standardgenetic methods, Jung et al. ((1994) supra) had previously localized thelocus on the long arm of chromosome 4.

Since the Rcg1 locus had previously been mapped to the long arm of maizechromosome 4, using the information on markers near the locus obtainedby Jung et al. (1994) supra, all available public and private simplesequence repeat (SSR) markers located in the region of the chromosomedesignated 4.06-4.08 were analyzed to determine if these markers werepolymorphic between the two near isogenic lines DE811 and DE811ASR(BC5). The DE811ASR (BC5) line was derived from the DE811ASR (BC3) linedescribed by Weldekidan and Hawk (1993), supra through two backcrossesto DE811 under selection for resistance to Cg, followed by 5 generationsof selfing and selection to obtain the BC5 line. The BC5 line wasbackcrossed twice more to DE811 to create the BC7 segregating populationused for fine mapping. In order to be able to conduct phenotypicevaluation on a family basis, BC7 individuals were selfed to createBC7S1 families.

From this analysis two SSR markers, PH1093 and UMC2041, were discoveredto be polymorphic. Using the publicly available inter-mated (Coe et al.(2002) Plant Physiol. 128:9-12; Gardiner, et al., (2004), PlantPhysiol., 134:1317-1326; Yim et al., (2002) Plant Physiol.130:1686-1696) B73×Mo17 (IBM) neighbors map (Lee et al. (2002) Plant MolBiol 48:453-61; Sharopova et al., (2002) Plant Mol Biol 48:463-81), thesequences of three nearby Restriction Fragment Length Polymorphism(RFLP) markers, CDO365, CSU166 and CDO127, were used to create fragmentlength polymorphic markers (hereafter designated FLPs). FLPs are markersthat can be assayed using gel electrophoresis or any similarhigh-resolution fragment separation method following a PCR reactionusing primers of a defined sequence. All three markers were found to bepolymorphic. The FLPs used in mapping the Rcg1 locus are summarized inTable 1. Any primers for the MZA FLPs shown on Table 1, which also havethe same MZA markers names shown on Table 2, will amplify a region ofthe FLP internal to the internal sequence shown on Table 2. Theannealing temperature for all the primers listed in Table 1 is 60° C.

In order to determine whether the presence of these three polymorphicFLPs and two polymorphic SSRs was associated with the resistantphenotype, indicating that the region carrying the Rcg1 locus waslocated on a chromosomal segment containing these three markers, a tablewas created in which the phenotypic status of 4784 individualsdetermined by field observation and the genotypic status relative toeach of the five markers, determined by fragment size analysis, wereentered. This data was submitted sequentially to the software programsJoinmap (Van Ooijen, et al., (2001), Plant Research International,Wageningen, the Netherlands) and Windows QTL Cartographer (Wang, et aL,(2004), (online, version 2.0 retrieved on Jun. 14, 2004 and version 2.5retrieved on Feb. 22, 2005); retrieved from the North Carolina StateUniversity Statistical Genetics and Bioinformatics website. The formerprogram determines the order of the markers along the chromosomalregion. The latter determines if a particular allele of a marker (aparticular form of the two polymorphic forms of the marker) issignificantly associated with the presence of the phenotype. Markers forwhich the presence of one or the other allele is more significantlyassociated with the resistant phenotype are more likely to be closer tothe gene responsible for the resistant phenotype. FIG. 3 depicts a graphproduced by Windows QTL Cartographer showing a statistical analysis ofthe chance (Y axis) that the locus responsible for the Cg resistancephenotype is located at a particular position along the chromosome (Xaxis) as defined by FLP markers.

From the integrated physical and genetic map as described by Fengler, etal., ((2004) Plant and Animal Genome XII Abstract Book, Page 192 (Posternumber P487), January 10-14, San Diego, Calif.) and Gardiner, (2004)supra, it was possible to identify two bacterial artificial chromosome(BAC) contigs, derived from a Mo17 BAC library, harboring the abovementioned genetic markers.

However, the two BAC contigs containing the markers flanking the regionof interest contained a gap of unknown size. In order to identifyfurther BACs to bridge this gap, a dense genetic map containing markers(Fengler, (2004) supra) with known positions on the physical map wasused to find additional markers genetically linked to markers previouslyidentified on the two BAC contigs. These additional markers in Table 2,were used to identify BAC contigs from a B73 BAC library which closedthe physical gap between the previously found Mo17-derived BAC contigs(Coe et al. (2002) supra; Gardiner (2004) supra; Yim et al. (2002)supra. Four markers, MZA11455, MZA6064, MZA2591 and MZA15842, were usedfor mapping purposes. In Table 2, “E” stands for “external” and “I”stands for “internal,” which respectively refer to the outer and innerprimers used during nested PCR. The external set is used in the firstround of PCR, after which the internal sequences are used for a secondround of PCR on the products of the first round. This increases thespecificity of the reaction. Upper case letters indicate portions of theprimer based on vector sequences, which are later used to sequence thePCR product. They are not maize sequences. For the forward internalnested MZA primers, the upper case portion of the sequence is SEQ ID NO:126, and for the reverse internal nested MZA primers, the upper caseportion is SEQ ID NO: 127. The sequences shown in Table 2 for theinternal forward MZA nested primers are therefore a combination of SEQID NO: 126 plus the SEQ ID NO: for each respective primer. Similarly,the sequences shown in Table 2 for the internal reverse MZA nestedprimers are a combination of SEQ ID NO: 127 plus the SEQ ID NO: for eachrespective primer. These combinations are indicated in the SEQ ID NO:column of Table 2. The annealing temperature for all the primers listedin Table 2 is 55° C. All markers set forth in Table 2 have shownpolymorphism within a diverse panel of corn germplasm, including MP305and the corn lines shown on Table 18.

The sequences of the ends of several of these BACs, as well as ESTsknown to be located on these BACs, were used in order to identify newmarkers with which to further narrow the range in which the locus waslocated. The further markers used for this purpose are designated FLP8,FLP27, FLP33, FLP41, FLP56 and FLP95 in Table 1. In a manner similar tothat described above, phenotype and genotypic correlations were made. Itwas determined that the locus was most likely located between FLP 8 andFLP 27 (See FIG. 3).

TABLE 1 Markers and primer pairs used in Examples 1, 4 and 5 Used inSEQ ID SEQ ID Example Name Forward NO: Reverse NO: 1, 4 FLP8CATGGAAGCCCCACAATAAC 24 ACATGGGTCCAAAGATCGAC 23 1, 4 FLP27AGCCCTATTTCCTGCTCCTG 26 GCATGCCCCATCTGGTATAG 25 1, 4 FLP33CTGTCGTTCGGTTTTGCTTC 28 GCATTCACATGTTCCTCACC 27 1, 4 FLP41TGTGTTCGCATCAAAGGTGT 30 CTGTAAGGCACCCGATGTTT 29 1, 4 FLP56GGTCTGGGAATGCTAAAGAGG 32 TGTCCAGGGTTACAGAAAACG 31 1, 4 FLP95ATTTCGACGGAGGGTTCTTC 33 GCAGCAGGAGGAGCTCATAG 34 4 FLP110ATGGAGGCTGCCCTGCTGAG 35 CGTATACCTCTCTGGCAAGGACGG 36 4 FLP111TTCCTGTTCGTCTGTATCTGATCCG 37 TTTGATTCCGGTCGAGTATAACCTG 38 4 FLP112GAAACTGCCTTCCCAGAAAACAATG 39 CAAGATCGGTGAAGTTGGTGCTTC 40 4 FLP113FATCACAGATGGGTCTCAAGGATTGC 41 4 FLPA1R TTCCAAGCAATTCACAGCTC 42 1, 5UMC1612 AGGTCCAGGTTACAGAGCAAGAGA 43 GCTAGTAGGTGCATGGTGGTTTCT 44 1, 4, 5UMC2041 CTACACAAGCATAGAGGCCTGGAG 45 CAGTACGAGACGATGGAGGACAT 46 1, 4CDO127 TGCTGTTGTTACTCGGGTTG 47 CTCTGCCTCAGCACAAATTC 48 1, 4, 5 PHI093AGTGCGTCAGCTTCATCGCCTACAAG 49 AGGCCATGCATGCTTGCAACAATGGATACA 50 1, 4CDO365 CTTCCAGAGGCAAAGCGTAG 51 TGTCACCCATGATCCAGTTG 52 1, 4, 5 CSU166TATTGTGCACGTCACCTTGG 53 GGGCAGACTTACTGCTGGAG 54 1, 4 UMC2285ATCTGCCTCCTTTTCCTTGG 55 AAGTAGCTGGGCTTGGAGGG 56 1, 4 MZA11455ACGAAGCAATTTCACCTTCC 57 TGTGGAACTAACCCTCAGCATAG 58 1 MZA6064CGAGAACCGGAGAAGAAGG 59 TTGGGCTGCTGTATTTTGTG 60 1, 4 MZA15842GACGCAGCTGTGAAGTTGG 61 CACCGGAATACCTTGACCAC 62 1, 5 UMC1086CATGAAAGTTTTCCTGTGCAGATT 63 GGGCAACTTTAGAGGTCGATTTATT 64 5 UMC1466GATCCACTAGGGTTTCGGGGT 65 CGAATAGTGGTCTCGCGTCTATCT 66 5 UMC1418GAGCCAAGAGCCAGAGCAAAG 67 TCACACACACACTACACTCGCAAT 68 5 BNLG2162CACCGGCATTCGATATCTTT 69 GTCTGCTGCTAGTGGTGGTG 70 5 CSU166AAATATCGGCTTTGGTCACG 71 TCGTCCTTCCTCAATTCGAC 72 5 UMC1051AATGATCGAAATGCCATTATTTGT 73 CTGATCTGACTAAGGCCATCAAAC 74 5 UMC2187ACCCAACAAGTCTTAATCGGGTTT 75 GTCCACCCTACCTCTCAACAAACA 76 5 UMC1371CATGTGAATGGAAGTGTCCCTTT 77 GCATCCTTTTCGTTTCAAATATGC 78 5 UMC1856AGATCTGTTTTGCTTTGCTCTGCT 79 CATGCCTTTATTCTCACACAAACG 80

TABLE 2 Nested MZA Primer Pairs Used in Example 1 SEQ ID SEQ ID NameForward NOs: Reverse NOs: MZA1215 E Agcccaattctgtagatccaa  81Tgcatgcaccggatccttc  82 MZA1215 I TGTAAAACGACGGCCAGTagcagcagacgatgcaaa126 + 83  GGAAACAGCTATGACCATGaggctggcggtggactt 127 + 84  ga ga MZA1216 ECcggcctacggcaacaagaa  85 agggtacggtgacccgaag  86 MZA1216 ITGTAAAACGACGGCCAGTttcgagacgctgtcgtac 126 + 87 GGAAACAGCTATGACCATGacgacgcatggcactag 127 + 88  ct cta MZA3434 ETgtaccgcgagaactcca  89 ttgcattcacatgttcctcac  90 MZA3434 ITGTAAAACGACGGCCAGTctactacgacggccgcta 126 + 91 GGAAACAGCTATGACCATGttgcagtagttttgtag 127 + 92  cagg MZA2591 EAgtaaataacagcattgacctc  93 tccaacggcggtcactcc  94 MZA2591 ITGTAAAACGACGGCCAGTctatataacagggccctg 126 + 95 GGAAACAGCTATGACCATGcacaaagcccacaagct 127 + 96  gaa aag MZA11123 EAccacaatctgaagcaagtag  97 cacagaaacatctggtgctg  98 MZA11123 ITGTAAAACGACGGCCAGTaaagaccaagaaatgcag 126 + 99 GGAAACAGCTATGACCATGagacatcacgtaacagt 127 + 100 tcc ttcc MZA15842 ECtcgattggcatacgcgata 101 ttccttctccacgcagttca 102 MZA15842 ITGTAAAACGACGGCCAGTagaaggtatttgccatgg 126 + 103GGAAACAGCTATGACCATGgtttcacttgctgaagg 127 + 104 ctta cagtc MZA11455 EGaccgatgaaggcaattgtga 105 accaaatagtcctagataatgg 106 MZA11455I ITGTAAAACGACGGCCAGTttcaaccttctgactgac 126 + 107GGAAACAGCTATGACCATGtaaacatagtcataaaa 127 + 108 acat attac MZA6064 ETcgaatgtattttttaatgcgg 109 atccacaatggcacttgggt 110 MZA6064 ITGTAAAACGACGGCCAGTcagctatttttgtcttct 126 + 111GGAAACAGCTATGACCATGggtcagattccaattcg 127 + 112 tcct gac MZA11394 ETcgtcctaacagcctgtgtt 113 gtccggatcaaatggatcgt 114 MZA11394 ITGTAAAACGACGGCCAGTaacagcctgtgttgaata 126 + 115GGAAACAGCTATGACCATGcgtgttccgtcgaggga 127 + 116 aggt gt MZA8761 ETtctttgattctactcttgagc 117 cttcatggacgcctgagatt 118 MZA8761 ITGTAAAACGACGGCCAGTtagagctttctgaactga 126 + 119GGAAACAGCTATGACCATGttggcatttagcttctc 127 + 120 tagc tcca MZA1851 EAtatattgcaccacttaaagcc 121 gggtgttatcacttgttctata 122 MZA1851 ITGTAAAACGACGGCCAGTtggagtccttgaccattt 126 + 123GGAAACAGCTATGACCATGtatatgcacttctagcg 127 + 124 gc agtat MZA16510 EAacaacaaggcgacggtgat 127 Tcatcttcgtcgtcctcatc 130 MZA16510 ITGTAAAACGACGGCCAGTgatcatcctgccggagtt 126 + 131GGAAACAGCTATGACCATGaaccgaaaacacaccc 127 + 132 tc MZA1719 Eccagcggtagattatatacag 133 cggtttggtctgatgaggc 134 MZA1719 ITGTAAAACGACGGCCAGTctcgggaaccttgttgg 126 + 135GGAAACAGCTATGACCATGtgaaatccagaacctcc 127 + 136 ga tttg

Example 2 Isolation of BAC Clones from the Resistant Lines andIdentification of Candidate Genes in the Region of the Rcg1 Locus

In order to isolate the gene responsible for the phenotype conferred bythe Rcg1 locus, BACs containing the region between the FLP 8 and FLP 27markers were isolated from a BAC library prepared from the resistantline DE811ASR (BC5). This library was prepared using standard techniquesfor the preparation of genomic DNA (Zhang et al. (1995) Plant Journal7:175-184) followed by partial digestion with HindIII and ligation ofsize selected fragments into a modified form of the commerciallyavailable vector pCC1 BAC™ (Epicentre, Madison, USA). Aftertransformation into EPI300™ E. coli cells following the vendorsinstructions (Epicentre, Madison, USA), 125,184 recombinant clones werearrayed into 326 384-well microtiter dishes. These clones were thengridded onto nylon filters (HYBOND® N+, Amersham Biosciences,Piscataway, USA).

The library was probed with overlapping oligonucleotide probes (overgoprobes; Ross et al. (1999) Screening large-insert libraries byhybridization, p. 5.6.1-5.6.52, In A. Boyl, ed. Current Protocols inHuman Genetics. Wiley, New York) designed on the basis of sequencesfound in the BAC sequences shown in the previous example to be presentbetween FLP8 and FLP27. BLAST™ search analyses were done to screen outrepeated sequences and identify unique sequences for probe design. Theposition and interspacing of the probes along the contig was verified byPCR. For each probe two 24-mer oligos self-complementary over 8 bp weredesigned. Their annealing resulted in a 40 bp overgo, whose two 16 bpoverhangs were filled in. The probes used in this way are presented inTable 4. Note that some of these probes were based on markers also usedin Example 1 and Table 1, but the exact sequences are different as theywere to be used as overgo probes rather than just PCR primers. Probesfor hybridization were prepared as described (Ross et al. (1999) supra),and the filters prepared by the gridding of the BAC library werehybridized and washed as described by (Ross et al. (1999) supra).Phosphorimager analysis was used for detection of hybridization signals.Thereafter, the membranes were stripped of probes by placing them in ajust-boiled solution of 0.1×SSC and 0.1% SDS and allowing them to coolto room temperature in the solution overnight.

BACs that gave a positive signal were isolated from the plates.Restriction mapping, PCR experiments with primers corresponding to themarkers previously used and sequences obtained from the ends of each BACwere used to determine the order of the BACs covering the region ofinterest. Four BACs that spanned the entire region were selected forsequencing. These BACs were sequenced using standard shotgun sequencingtechniques and the sequences assembled using the Phred/Phrap/Consedsoftware package (Ewing et al. (1998) Genome Research, 8:175-185).

After assembly, the sequences thought to be in the region closest to thelocus on the basis of the mapping data were annotated, meaning thatpossible gene-encoding regions and regions representing repetitiveelements were deduced. Gene encoding (genic) regions were sought usingthe FGENESH™ software package (Softberry, Mount Kisco, N.Y., USA).FGENESH™ predicted a portion of a protein, that when compared usingBLAST® (BLASTx/nr), displayed partial homology at the amino acid levelto a portion of a rice protein that was annotated as encoding for aprotein that confers disease resistance in rice. The portion of themaize sequence that displayed homology to this protein fell at the endof a contiguous stretch of BAC consensus sequence and appeared to betruncated. In order to obtain the full representation of the gene in themaize BAC, the rice amino acid sequence was used in a tBLASTn analysisagainst all other consensus sequences from the same maize BAC clone.This resulted in the identification of a consensus sequence representingthe 3′ end of the maize gene. However, the center portion of the genewas not represented in the sequences so obtained. PCR primers weredesigned based on the 5′ and 3′ regions of the putative gene and used ina PCR experiment with DNA from the original maize BAC as a template. Thesequence of the resulting PCR product contained sequence bridging the 5′and 3′ fragments previously isolated.

DE811ASR (BC5) has been deposited with the ATCC, and the methodsdescribed herein may be used to obtain a BAC clone comprising the Rcg1locus. As shown in FIG. 9( a), the DE811ASR (BC5) chromosomal intervalwith the Rcg1 locus is non-colinear with the corresponding region of B73and Mo17 (See FIGS. 9 and 22), as determined by the analysis of BAClibraries.

Using common sequence that hybridize to BACs in the Mo17 and the B73 BAClibraries, the corresponding BACs from both libraries were lined up in atiling path as shown in FIG. 22. The B73 BACs in FIG. 22 were givenshorter names for the purposes of the figure. Table 3, below, shows theBAC ID for each BAC designation indicated on FIG. 22. The public B73BACs, c0113f01 and c0117e18 are directly north and south, respectively,of the Rcg1 locus indel region, with the deletion occurring in B73.Information about these two BACs can be viewed on several websitesincluding the maize GDB website (maizegdb.org), the Gramene website(gramene.org) and the maize genome database of the Arizona GenomicsInstitute (genome.arizona.edu). The Arizona Genomics Institute websitealso provides the Maize Agarose FPC Map, version Jul. 19, 2005, whichidentifies BACs contiguous with c0113f01 and c0117e18. By searching onthose databases, a multitude of BACs were identified that form a contigof the regions flanking the Rcg1 locus. Thus, the precise location ofthe Rcg1 locus and Rcg1 gene have now been identified on both the maizegenetic and physical map. See FIGS. 7( a, b) and 22.

TABLE 3 BAC designations in FIG. 22, which were part of either the 187contig (B73a through B73p) or 188 contig (B73q through B73af) of B73asshown on the Arizona Genomics Institute website mentioned above. B73 BACdesignation in FIG. 22 B73 BAC ID B73a c0100m06 B73b b0050k15 B73cc0127n01 B73d c0449o09 B73e c0046c06 B73f c0212g06 B73g c0153l14 B73hc0105c14 B73i b0502a04 B73j b0239l06 B73k b0171g07 B73l c0273k24 B73mc0113f01 B73n c0117e18 B73o c0119n15 B73p b0369n20 B73q b0031c17 B73rc0081g12 B73s c0303g03 B73t c0222i18 B73u c0428j12 B73v c0314e18 B73wc0150j16 B73x b0085n01 B73y c0040c01 B73z c0018f13 B73aa c0091e23 B73abb0100g11 B73ac c0177e03 B73ad b0264h08 B73ae c0410a17 B73af c0012f18

The complete sequence of the putative gene is set forth in SEQ ID NO: 1.The gene contains one intron, from nucleotide 950 to nucleotide 1452 ofSEQ ID NO: 1. Reverse transcriptase-PCR using RNA prepared from DE811ASR(BC5) plants was used to determine the borders of the intron. Theprotein coding sequence of the gene is set forth in SEQ ID NO: 2, andthe amino acid translation is set forth in SEQ ID NO 3. The predictedprotein has a molecular weight of 110.76 kD.

The amino end from approximately amino acids 157 to 404 has homology toso-called nucleotide binding sites (NBS). There is a region with loosehomology to LRR domains located approximately from amino acids 528 to846. However, unlike previously studied NBS-LRR proteins, the leucinerich region lacks the systematic repetitive nature (Lxx) found in moreclassical LRR domains and in particular having no instances of theconsensus sequences described by Wang et al. ((1999), Plant J. 19:55-64)or Bryan et al. ((2000), Plant Cell 12:2033-2045). The gene has loosehomology with a family of rice genes and a barley gene as shown in FIG.2 (a, b and c). Most of the homology is at the amino terminal end of theprotein; the carboxyl end is quite distinct. This is demonstrated by theuse of bold type, in FIGS. 2 (a, b and c), which are amino acidsidentical to the gene of the embodiments, while those which arenon-identical are not shown in bold type.

TABLE 4 Oligonucleotides annealed to synthesize overgo probes AssociatedForward SEQ Reverse SEQ Genetic oligonucleotide ID oligonucleotide IDmarker sequence NO: sequence NO: FLP8 cagggcctacttggt 4 gggtactacactagc5 ttagtaata ctattacta None cggttacaaggtcta 6 gtcaaacagatagcc 7 cccaatctggcagattgg FLP33/PHI93 tacaaaactactgca 8 cctcaccccaagtat 9 acgcctataatataggcg FLP27 cattggacctcttcc 10 tccttgagtccagtg 11 ccactaagactcttagtg None gaaactaggcgcgtc 12 aaggcagccactgaa 13 aggttttat aataaaacc

Example 3 Comparison of Genetic Structure in the Region of the Rcg1Locus Between Resistant and Susceptible Lines and Expression Profiles ofCandidate Genes Found in that Region Between Resistant and SusceptibleLines

Having found a candidate gene in the region genetically defined to carrythe locus responsible for the resistance to anthracnose phenotype,efforts were undertaken first to determine if there might be other genespresent in the region and second to determine if the expression patternsof the candidate gene were consistent with its putative role. Fu andDooner ((2002), Proc Natl Acad Sci 99:9573-9578) and Brunner et al.((2005), Plant Cell 17:343-360) have demonstrated that different corninbred lines may have significant rearrangements and lack of colinearitywith respect to each other. Comparison of such genomes over largerregions can thus be complex. Such a comparison of the genomes of Mo17(Missouri 17) and DE811ASR (BC5) revealed that in the region where thecandidate gene is found in DE811ASR (BC5), a large insertion relative toMo17 is present. Regions within and surrounding the insertion weresequenced and scanned for possible genes. A gene encoding a subunit ofRibulose bisphosphate carboxylase (Rubisco, a protein involved in carbonfixation after photosynthesis whose gene is present in multiple copiesin the corn genome) was found in both the DE811ASR (BC5) and Mo17genomes, just downstream of the position of the Rcg1 gene. A pseudogene(a gene rendered nonfunctional due to mutations disrupting the codingsequence) related to a vegetative storage protein was found, presentonly in the DE811ASR (BC5) genome some distance upstream of the Rcg1gene. The only structurally intact gene likely to encode a protein witha function likely to be related to disease resistance was the Rcg1 geneisolated in the previous example. Other genes equally unlikely to beinvolved in disease resistance were located at a greater distance fromthe most likely position of the locus, as well as a large number ofrepetitive sequences.

In order to determine if and where the Rcg1 gene was transcribed, twotechniques were used. First, the RNA profiles of resistant andsusceptible plant materials were surveyed using Massively ParallelSignature Sequencing (MPSS™; Lynx Therapeutics, Berkeley, USA). Briefly,cDNA libraries were constructed and immobilized on microbeads asdescribed (Brenner, S. et al. (2000) Nat. Biotechnol. 18(6): 630-634).The construction of the library on a solid support allows the library tobe arrayed in a monolayer and thousands of clones to be subjected tonucleotide sequence analysis in parallel. The analysis results in a“signature” 17-mer sequence whose frequency of occurrence isproportional to the abundance of that transcript in the plant tissue.cDNA derived from RNA prepared from DE811ASR(BC5) and from DE811(control line, susceptible to Cg) was subjected to MPSS™ analysis.Bioinformatic inspection of the resulting signatures showed that asignature sequence, referred to herein as Lynx19, (SEQ ID NO: 19) waspresent at 43 parts per million (ppm) in RNA samples from DE811ASR (BC5)uninfected stalks and at 65 ppm in infected, resistant stalks 9 dayspost inoculation (DPI) with Cg. This signature sequence was not detectedin cDNA libraries of uninfected or Cg-infected stalks of the susceptiblecorn line DE811. An analysis of the sequence of Rcg1 indicates that the17-mer tag is present at nucleotides 3945 to 3961 of SEQ ID NO: 1 in theputative 3′ untranslated region of the gene.

Further proof that Rcg1 is exclusively expressed in corn lines that arederived from MP305 and resistant to anthracnose stalk rot was obtainedby RT-PCR experiments. Total RNA was isolated from uninfected andCg-infected stalks of resistant (DE811ASR1 (BC5)) and susceptible(DE811) corn lines using RNA STAT-60™ (Iso-Tex Diagnostics, Friendswood,Tex., USA). Total RNA (250 ng) from 0, 3, 6, 9, and 13 DPI resistant andsusceptible samples was copied into cDNA and amplified using a GENEAMP®RNA-PCR kit (Applied Biosystems, Foster City, Calif., USA). The cDNAsynthesis reaction was assembled according to the kit protocol usingrandom hexamers as primers and incubated at 42° C. for 45 minutes. ForPCR, KEB131 (SEQ ID NO: 20) and KEB138 (SEQ ID NO: 21), both designedfrom the putative 3′ untranslated sequence of Rcg1, were used as theupstream and downstream primers, respectively. The cDNA was amplifiedfor 30 cycles consisting of 1 minute at 94° C., 2 minutes at 50° C. and3 minutes at 72° C. followed by a 7 minute extension at 72° C. As shownin FIG. 4, agarose gel electrophoresis of an aliquot of the RT-PCRsrevealed the presence of a 260 bp band present in the samples derivedfrom both infected and uninfected resistant plants but absent fromsusceptible samples. DNA sequence analysis confirmed that this fragmentcorresponded to nt 3625 to 3884 of the Rcg1 sequence consistent with theamplification product predicted from primers KEB131 and KEB138.

Example 4 Isolation of Lines Containing Mu Insertions in the CandidateGene

One method to determine if a gene is responsible for a phenotype is todisrupt the gene genetically through the insertion of a transpositionelement (so-called transposon tagging) and then determine if therelevant phenotype of the plant is altered, in this case from resistantto Cg to susceptible to Cg. In corn this can be done using the mutator(Mu) element (Walbot, V. (1992) Annu. Rev. Plant Physiol. Plant Mol.Biol. 43:49-82). The basic strategy, outlined in FIG. 5, was tointroduce active mutator elements into lines carrying the resistancegene, isolating plants homozygous for the resistance gene by assayingassociated DNA markers as well as resistance to Cg by inoculation withCg, then crossing those homozygous plants with a susceptible “tester”line. If the resistance gene is dominant, in principle all the resultingprogeny would be resistant but heterozygous for the gene. However, if aMu element inserted into the resistance gene in a way that disrupted itsfunction, that individual would be susceptible to Cg. The disrupted genecan then be isolated and characterized.

MP305 was crossed with fifteen diverse mutator stocks (lines carryingactive mutator elements). The resulting F1 s were inter-mated (crossedwith each other) in all possible combinations. To track the chromosomalregion 4L on which the resistance locus was known to reside (seeExample 1) a variety of DNA markers known to be in the vicinity of thelocus from the work described in Example 1 were selected and used on theMu-tagged materials. About 1500 progeny plants from the inter-matingprocess were examined for resistance to Cg and for the presence of thesemarkers. Analysis of the markers was done using either Southern blots(Botstein et al., (1980) Am. J. Hum. Gen. 32:314-331) for RFLP markersor by PCR for FLP markers as described in Example 1. Plants that werehomozygous for all the markers tested and resistant to Cg were selectedand test crossed with susceptible tester lines (A63, EH6WA and EF09B).About 16,000 test cross seeds generated from these homozygous andresistant plants were then planted and were used as female parents(meaning the pollen producing tassels were removed) and crossed with thesusceptible tester lines used as males. All the female plants werescreened for susceptibility to Cg. More than ten susceptible plants(putative knockout mutants) were identified. The open pollinated seedfrom each of these susceptible plants was harvested, along with eightresistant siblings as controls.

DNA from a pool of 24 seedlings (grown in paper towels) from each of theputative knockouts and the control resistant siblings was extracted.This DNA was used as template for amplifying the flanking sequence fromthe site of Mu-insertion using gene-specific primers in combination witha consensus primer designed from the terminal inverted repeats (TIR)from the Mutator element sequence (SEQ ID NO: 125). In other words, PCRproducts would only be observed if a Mu element had inserted into thecandidate gene isolated in Example 2. The primers FLP110F, FLP110R,FLP111F, FLP111R, FLP112F, FLP112R, FLP113F, and FLPA1R were used as thegene-specific primers (See Table 1). PCR amplified products were blottedonto nylon membranes and hybridized with a DNA probe from the candidategene isolated in Example 2. PCR products that showed stronghybridization were excised from the gel, purified, cloned and sequenced.The resulting sequences were analyzed by aligning with sequences fromthe candidate gene and Mu-TIR. Mutator elements cause a direct 9 bpduplication at the site of insertion. Based on the flanking sequenceinformation and a direct 9 bp duplication, four independent insertionswere identified in exon 1 of the candidate gene (FIG. 5). One insertion(m177) was detected approximately 97 bp upstream of the initiationcodon, in the 5′ untranslated region of the gene. One common insertionevent, 270 bp downstream of the initiation codon, was detected in threesusceptible plants: m164, m159, and m179. The m171 susceptible plant wasfound to contain two Mu-insertions, 556 bp and 286 bp downstream of theinitiation codon. When Southern blots were carried out using the exon1region of the gene as a DNA probe, the modified hybridization patternobserved further confirmed these results.

This and the preceding examples may be summarized as follows. Theearlier work cited in Example 1 showed that a previously observed locusconferring resistance to Cg was localized on the long arm of maizechromosome 4. The nature of this locus, its exact location or thegene(s) encoded by it were completely unknown. The work done in Example1 demonstrates that the locus can be mapped to a very small region ofthe long arm of chromosome 4. Example 2 demonstrates that there is agene to be found in this chromosomal region encoding a novel form of anNBS-LRR protein, a family of proteins known to be involved in resistanceto pathogens but which vary widely in their sequence and specificity ofresistance. Example 3 shows that this gene is present only in theresistant line, not the isogenic susceptible line, and that transcriptscorresponding to this gene are found in the resistant line, indicatingthat the gene is expressed, and these transcripts are found only in theresistant line. Example 4 demonstrates that in four independentlyisolated Mu insertion events, when the gene is disrupted by insertion ofa Mu element, the phenotype of these plants is changed from resistant tosusceptible to Cg. Taken together, these data provide overwhelmingevidence that the subject of the embodiments of this invention is a genenecessary in enhancing or conferring Cg resistance in corn plants.

Example 5 Backcrossing of the Rcg1 Locus into Susceptible Lines

An Rcg1 locus introgression of an inbred was made to confirm that theRcg1 locus could be successfully backcrossed into inbreds, and thathybrids produced with the inbred line with the Rcg1 locus would haveenhanced or conferred Cg resistance. DE811ASR (BC5) was also developedand used as an improved donor source for introgression of the Rcg1locus. Next, several additional inbreds were utilized as recurrentparents in order to use the marker assisted breeding methods describedherein to efficiently introgress the Rcg1 locus into a variety of inbredand hybrid genetic backgrounds, thereby enhancing or conferringresistance to Cg. Each of these examples are discussed in more detailbelow.

Proof of Concept (PH09B)

MP305 is a white kernel color inbred line with strong resistance to Cg,but its late flowering, poor yield and weak agronomic characteristicsmake it a poor donor parent in the absence of the use of the markerassisted breeding methods described herein. A molecular marker profileof MP305 is provided in Table 5b. Primers used for the SSRs reported inthe table can be constructed from publicly available sequences found inthe Maize GDB on the World Wide Web at maizegdb.org (sponsored by theUSDA Agricultural Research Service), in Sharopova et al. (Plant Mol.Biol. 48(5-6):463-481), and/or in Lee et al. (Plant Mol. Biol. 48(5-6);453-461). UMC15a is an RFLP marker, and the score reported is based onEcoR1 restriction.

To demonstrate the phenotypic value of the Rcg1 locus, the locus wasfirst introgressed into line PH09B (U.S. Pat. No. 5,859,354) through tothe BC3 stage as follows. The F1 population derived from the crossbetween MP305 and line PH09B was backcrossed once more to line PH09B,resulting in a BC1 population. Seedlings were planted out andbackcrossed again to line PH09B to develop a BC2 population. DNA wasprepared from leaf punches of BC2 families. To determine which BC2families to plant for further backcrosses, genotyping was carried out onDNA from BC2 families using primers for markers flanking the region ofinterest, UMC2041, PHI093 and CSU166 (See Table 1). Seeds from BC2families were planted and individual plants were genotyped again for thepresence of the MP305 version of that region of the chromosome using thesame three markers noted above. Positive plants were backcrossed to linePH09B once more to develop BC3 populations. Seed from these BC3populations was planted and plants were selfed to obtain BC3S1 familiessegregating for the region of interest as well as BC3S1 families missingthe region of interest. These families were used for phenotypiccomparison (BC3S1 segregating versus BC3S1 without the region ofinterest).

In order to observe the performance of the Rcg1 gene in a heterozygoussituation such as would be found in a commercial hybrid, appropriatetestcrosses were made. Specifically, BC3S1 families segregating for theregion of interest were planted and individual BC3S1 plants weregenotyped. Plants homozygous for the Rcg1 gene as well as plantshomozygous for the null allele (lacking the gene on both chromosomes)within each family were used to make testcrosses with inbreds PH2EJ(U.S. Pat. No. 6,333,453), PH2NO (U.S. Pat. No. 6,124,533), PH4CV (U.S.Pat. No. 6,897,363) and PH8CW (U.S. Pat. No. 6,784,349).

In the case of both the BC3S1 lines and the hybrids, the observedphenotypic differences indicated significant improvement for ASRresistance in lines and hybrids containing the region carrying Rcg1. Theeffect of the introgressed Rcg1 locus in the BC3S1 families and thederived testcross hybrids resulted in an improvement in terms of boththe number of internodes infected and the number of internodes infectedat more than 75%. The scores, using a visual scoring system commonlyused by plant breeders, are shown in Table 5a below. The data clearlydemonstrate that using crossing techniques to move the gene of theembodiments into other lines genetically competent to use the generesult in enhanced resistance to Cg.

TABLE 5a Effect of the introgressed Rcg1 region on degree of resistanceto anthracnose stalk rot in BC3S1 families and derived test crosses.Number of Number of internodes internodes >75% Rcg1 infected infectedBC3S1 Absent 3.1 2.4 Present 2.3 1.5 Difference 0.8 0.9 PH2EJ Absent 2.61.5 Present 2.1 0.9 Difference 0.5 0.6 PH2NO Absent 3.0 2.1 Present 2.41.3 Difference 0.6 0.8 PH4CV Absent 2.8 1.8 Present 2.2 1.0 Difference0.6 0.8 PH8CW Absent 2.9 1.7 Present 2.3 0.8 Difference 0.6 0.9

TABLE 5b Molecular marker profile of MP305 Marker Base Pair Base PairName Weight Bin Marker Name Weight Bin phi295450 191.1 4.01 umc1667154.65 4.08 phi213984 302.23 4.01 phi438301 212.76 4.05 phi096 235.074.04 umc1808 106.67 4.08 mmc0471 241.6 4.04 umc1043 199.6 4.07 umc196965.01 4.05 umc1871 148.48 4.08 umc1662 116.14 4.05 dupssr28 100.64 4.08umc2061 125.34 4.05 umc1466 110.91 4.08 phi079 185.76 4.05 umc1418153.12 4.08 bnlg1937 235.87 4.05 umc1899 111.81 4.08 umc1382 153.7 4.05bnlg2162 144.98 4.08 bnlg1217 194.36 4.05 umc2041 165.17 4.08 umc1390133.46 4.05 umc2285 156 4.08 bnlg1265 221.83 4.05 umc1086 95.57 4.08umc1303 127.2 4.05 umc1612 108.54 4.08 bnlg252 167.85 4.06 umc15a approx10 kb 4.08 with EcoRl restriction umc1895 142 4.05 cdo365 411.5 4.08umc1175 279.6 4.05 umc1051 125.9 4.08 umc1317 110.12 4.05 umc2187 84.944.08 umc1548 159.52 4.05 umc1371 120.6 4.08 umc1451 110.69 4.05 umc1132132.14 4.08 umc1896 87.89 4.05 umc1856 156.88 4.08 umc1511 166.43 4.05umc2153 131.97 4.08 umc1851 114.13 4.05 umc2200 151 4.08 umc1791 153.234.05 phi066 160 4.08 bnlg1755 216.93 4.05 umc1039 222.7 4.08 umc170294.8 4.05 umc2139 134.2 4.09 umc1346 96.39 4.05 umc1559 141.09 4.09umc1142 146.98 4.05 umc1999 131.55 4.09 mmc0371 230.82 4.06 umc1820138.94 4.09 umc1945 113.52 4.06 umc1173 168.02 4.09 umc1093 222.7 4.06umc1650 139.84 4.09 umc2027 111 4.06 umc1328 161.33 4.09 bnlg1621 184.114.06 umc1740 98.2 4.09 umc1299 144.46 4.06 umc1643 145.23 4.09 umc1869154.39 4.06 umc1989 100.5 4.09 bnlg2291 201.5 4.06 umc1284 144.39 4.09bnlg1784 237.23 4.07 umc1574 155.11 4.09 dupssr34 326.01 4.07 umc2137158.1 4.08 umc1651 99.59 4.07 umc1101 160.12 4.09 umc2038 122.19 4.07umc2046 115.82 4.09 umc1847 160.17 4.07 phi314704 143.54 4.09 umc1620148.2 4.07 bnlg1890 251.68 4.11 umc1194 162.29 4.07 phi076 158.05 4.11DE811ASR(BC5) as Most Improved Donor for Use in Backcrossing

Although MP305 was utilized in the above experiment, as is illustratedin FIG. 8( a), DE811ASR(BC5) retains a smaller MP305 chromosomalinterval with the Rcg1 locus than DE811ASR(BC3) (and of course MP305 aswell), and therefore is particularly useful as a donor source for theRcg1 gene. The shortened chromosomal interval from the DE811ASR(BC5)source has been shown to be associated with an improved agronomicphenotype. Twenty two plants from the DE811ASR(BC3) derived line, 20plants from the DE811ASR(BC5) derived line, five DE811 plants and fiveMP305 plants were grown in a greenhouse from November 2005 through March2006 and data were taken for plant height and ear height; dates when 50%of the plants shed pollen (midshed), when 50% of the plants had visualear shoots (midves) and when 50% of the plants had silks protruding fromthe earshoots (midslk); and kernel color was observed. On average, theDE811ASR(BC5) line was shorter than DE811ASR(BC3) (293 cm vs 345 cm) andthe location of the ear was lower in the DE811ASR(BC5) than in theDE811ASR(BC3) (146 cm vs 183 cm), both of which are positive traits interms of elite variety development. DE811ASR(BC5) was earlier formidshed, midves and midslk compared to DE811ASR(BC3). Midshed wasapproximately 1 day earlier, midves was approximately 6 days earlier andmidslk was approximately 3 days earlier for DE811ASR(BC5) compared toDE811ASR(BC3). Kernels of DE811ASR(BC5) had a yellowish-brown (bronze)color whereas kernels of DE811ASR(BC3) had a pale yellow cap. Dates formidshed, midves and midslk were similar for DE811ASR(BC5) and DE811,whereas MP305 was approximately 11 days later for midshed and did notproduce 50% visual ear shoots, nor 50% silks during the growing period.While these data are based on only a few plants for DE811 and MP305, andears were not produced on those few lines, these greenhouse resultsresemble observations of these lines in the field. These data indicatethat DE811ASR(BC5) resembles the DE811 recurrent parent much moreclosely than DE811ASR(BC3). Thus, DE811ASR(BC5) is an excellent initialdonor source for the Rcg1 locus and the Rcg1 gene, both genotypicallyand phenotypically. In addition, DE811ASR(BC5) is particularly usefulwhen introgressing the Rcg1 locus into germplasm with similar adaptationto DE811.

DE811 was developed by J. Hawk (Hawk, J. A. (1985). Crop Science Vol 25:p 716) and has been described as a yellow dent inbred line thatoriginated from selfing and selection for six generations in a pedigreeprogram out of a cross of B68 to an inbred derived from [B37Ht×(C103×Mp3204 double cross) sel.]. DE811 silked 1 to 2 days later thanB73 in tests in Delaware, but 4 days later than B73 at Missouri. Limitedyield trials indicate that DE811 has satisfactory combining ability. Itis a good silker (forms good silks, a component of the maize femaleflower important for fertility) and pollen shedder and can be crossed toearlier maturity germplasm for Northern US adaptation and to latermaturity germplasm for Southern US adaptation. Thus, DE811ASR (BC5), incombination with the markers and breeding methods disclosed herein, isuseful as an initial donor source for introgressing the Rcg1 gene into awide variety of germplasm, including germplasm adapted to all of theregions in the US where Cg is present.

Creation of Inbred Rcg1 Locus Conversions

Following the tests for successful Rcg1 locus introgression in PH09Bdescribed above, additional Rcg1 locus conversions were carried out onother inbred lines. The first series had 5 backcrosses, with MP305 andDE811ASR(BC5) as donors. For the second series of backcrosses, molecularmarkers were used to reduce the chromosome interval in the BC5conversions from the first series. These BC5 conversions were selectedfor crossovers below the Rcg1 gene. Those selected plants were thenbackcrossed to create the BC6 generation. Plants with crossovers abovethe gene were selected in the BC6 generation.

First Series of Backcrosses

In the first series, DE811ASR(BC5) was used as the primary donor source,but parallel introgressions were also made to the same inbreds usingMP305 as a donor source. These data, described in more detail below,show that while DE811ASR(BC5) is the preferred donor in many situations,MP305 can also be effectively used with the marker assisted breedingmethods of the embodiments taught herein.

Elite inbred lines primarily adapted to North American growingconditions were selected for use as recurrent parents. The inbreds linesinitially selected for use as recurrent parents were lines PH0R8 (U.S.Pat. No. 6,717,036), PH7CH (U.S. Pat. No. 6,730,835), PH705 (U.S. Pat.No. 690,325), PH5W4 (U.S. Pat. No. 6,717,040), PH51K (U.S. Pat. No.6,881,881) and PH87P (U.S. Pat. No. 6,888,051). Each of these lines wascrossed with DE811ASR (BC5) as well as with MP305. The F1 generationderived from each of these crosses was backcrossed once more to therespective inbred line, resulting in a first backcross (the recurrentparent BC1) generation. Seedlings were planted out and DNA was preparedfrom leaf punches. PCR reactions were carried out using primers formarkers flanking the region of interest; UMC1466, UMC1418, BNLG2162,UMC1086, UMC2041, UMC1612, CSU166, UMC1051, UMC2187, UMC1371, andUMC1856 were used in the early BC rounds (See Table 1) while in later BCrounds, UMC1418, BNLG2162, UMC1051, UMC2041, UMC2187, UMC1371 andUMC1856 were used. Seedlings whose PCR reactions gave a positive result(meaning that the MP305 derived Rcg1 locus was present) were thenfurther backcrossed to the respective inbred lines to make a BC2. Thisprocedure, called “genotyping”, identifies the genetic composition of aplant at the site of a particular marker. These steps were repeated forthe recurrent parent BC3, BC4 and BC5 development. Analysis shows that,after five backcrosses, these lines retained a significantly truncatedchromosomal interval comprising the Rcg1 locus, and, based on visualobservations, no indication of negative effects resulting from thepresence of the Rcg1 locus was observed.

Recurrent parent selection was also carried out by selecting the plantsmost phenotypically like the recurrent parent. Using these genotypic andphenotypic methods, high quality conversions were selected with a highpercentage of recurrent parent across the whole genome.

This example also illustrates that flanking markers are not usedexclusively to select either for or away from the Rcg1 gene. Seedlingswhose PCR reactions gave a positive result (meaning that the MP305derived Rcg1 locus was present) were then further backcrossed to therespective inbred lines to make the final backcross (the recurrentparent BC5 generation) in this first series. Where the closest flankingpolymorphic markers determined that the gene was present, the next setof double flanking polymorphic markers more distal to the gene were usedfor recurrent parent selection. Thus, the use of markers flanking theRcg1 gene or Rcg1 locus serves to illuminate the recombination occurringin the region.

Second Series of Backcrossing

The inbred Rcg1 locus conversions made using the SSRs flanking the Rcg1locus in the first series of backcrossing were then used as donors in asuccessive round of backcrossing. For this series of backcrossing, SNPmarkers were developed for the Rcg1 gene that enabled marker assistedselection in a high throughput manner, as described in Example 15, toselect for the Rcg1 gene. SNP markers were also designed in the regionaround the Rcg1 locus, allowing flanking markers to be used to selectaway from the MP305 chromosomal interval surrounding the Rcg1 locus, andto select for the recurrent parent genotype, thereby greatly reducinglinkage drag. It is only through physically mapping and cloning the genethat such precise marker-assisted recurrent parent selection ispossible.

First, the recurrent parent BC5 plants resulting from the first seriesof backcrossing were re-screened with the more precise marker set, andrecombination was selected for south of the Rcg1 gene. Flanking markerstightly linked to the Rcg1 gene (MZA8761, MZA1851, UMC1051, and UMC2187)were used to select for recurrent parent to the south of the gene insmall population sizes of approximately 40 progeny. (See FIG. 8( a-b)).These progeny were then analyzed using the FLP markers disclosed herein,to more precisely determine the point of recombination. This data showedthat some progeny were selected with recurrent parent genome less than 1cM (based on IBM2 Neighbors genetic map distances) south of the Rcg1gene, as shown in FIG. 8( b). Other progeny had recurrent parent genomeless than 4 cM south of the Rcg1 gene. These marker-selected BC5conversions were then used as donors, and crossed to near-isogeniccounterparts of PH705, PH5W4, PH51K and PH87P as the recurrent parentsto give a BC6 population. Markers in the Rcg1 gene were again used toselect for Rcg1, with flanking markers to the north of Rcg1 this timebeing used to select for recurrent parent. In this round of selections,recombinations were detected in each population between Rcg1 and themarker MZA15842. The position of MZA15842 on the IBM2 Neighbors geneticmap can be extrapolated from its position on the high resolution mapshown in FIG. 7( b), map B, using regression relative to the flankingmarkers UMC2285 and PH1093. This placed MZA15842 at 520.5 cM on the IBM2Neighbors genetic map. Therefore, as shown in FIG. 8( b), in two roundsof backcrossing, the donor genome was reduced to a segment of less than6 cM in each population, or less than 0.8% of chromosome 4, based on theIBM2 Neighbors genetic map distances, and in some progeny the segmentwas less than 2.1 cM, or less than 0.25% of chromosome 4. Forcomparison, the MP305 chromosomal interval with the Rcg1 locus inDE811ASR (BC3) was 131 cM, or approximately 16% of chromosome 4, basedon the IBM2 Neighbors genetic map distances. It is only throughphysically mapping and cloning the gene that such precise and efficientmarker-assisted recurrent parent selection is possible.

Further Analysis

Therefore, as a result of fine mapping the location of the Rcg1 gene,one may utilize any two flanking markers that are genetically linkedwith the Rcg1 gene to select for a small chromosomal region withcrossovers both north and south of the Rcg1 gene. This has the benefitof reducing linkage drag, which can be a confounding factor when tryingto introgress a specific gene from non-adapted germplasm, such as MP305,into elite germplasm, such as the inbred lines noted above. FIGS. 7 and22, and Table 16 show many combinations of markers flanking the Rcg1gene and locus that may be used for this purpose. Some specific flankingmarkers that may be used for selecting truncated chromosomal intervalsthat include the Rcg1 gene or locus are UMC2285 and UMC15a, UMC2285 andUMC2187, UMC1086 and UMC2200, UMC2041 and UMC2200, UMC2041 and PH1093,MZA11455 and UMC15a, MZA11455 and MZA3434, MZA15842 and MZA3434, andFLP8 and FLP33. Optionally, on or within each of these chromosomalintervals, one could utilize at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16 or more markers in order to locate the recombinationevent and select for the Rcg1 gene or Rcg1 locus with the maximum amountof recurrent parent genotype. Further, one may have at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more markers between the northend of such chromosomal interval and the top of the Rcg1 gene and/orbetween the south end of such chromosomal interval and the bottom of theRcg1 gene.

It is advantageous to have closely linked flanking markers for selectionof a gene, and highly advantageous to have markers within the geneitself. This is an improvement over the use of a single marker ordistant flanking markers, since with a single marker or with distantflanking markers the linkage associated with Rcg1 may be broken, and byselecting for such markers one is more likely to inadvertently selectfor plants without the Rcg1 gene. Since marker assisted selection isoften used instead of phenotypic selection once the marker-traitassociation has been confirmed, the unfortunate result of such a mistakewould be to select plants that are not resistant to Cg and to discardplants that are resistant to Cg. In this regard, markers within the Rcg1gene are particularly useful, since they will, by definition, remainlinked with resistance to Cg as enhanced or conferred by the gene.Further, markers within the Rcg1 locus are just as useful for a similarreason. Due to their very close proximity to the Rcg1 gene they arehighly likely to remain linked with the Rcg1 gene. Once introgressedwith the Rcg1 gene, such elite inbreds may be used both for hybrid seedproduction and as a donor source for further introgression of the Rcg1gene into other inbred lines.

Thus, the data clearly shows that inbred progeny converted by usingDE811ASR(BC5) as a donor source retain the truncated MP305 chromosomalinterval. The inbreds comprising the truncated MP305 chromosomalinterval are very useful as donor sources themselves, and there is noneed to revert to DE811ASR(BC5) as a donor source. By using markerassisted breeding as described herein, the truncated MP305 chromosomalinterval can be further reduced in size as necessary without concern forlosing the linkage between the markers and the Rcg1 gene.Phenotypically, a reduced chromosomal interval is associated withimproved agronomic performance, as was demonstrated for DE811ASR(BC5)versus DE811ASR(BC3) described above.

Example 6 Constructs for the Transgenic Expression of Rcg1

The Rcg1 gene was expressed as a transgene to determine if this genealone was sufficient, as well as necessary, for the phenotype conferredby the Rcg1 locus. This was done using two different constructs,differing chiefly in the promoter used.

Example 6a

In this example, the Rcg1 gene was expressed using its own promoter. Theupstream region of the Rcg1 gene was sequenced using the same BACs thatin Example 2 provided the sequences of the protein-coding section of thegene. The sequence of 1684 bp 5′ to the ATG is set forth in SEQ ID NO:24.

In order to transform the complete Rcg1 gene, including the promoter andprotein encoding region, a 5910 bp fragment extending from position41268 through position 47176 in SEQ ID NO: 137 was amplified by PCRusing a BAC clone as template DNA. To enable cloning using the GATEWAY®cloning system (Invitrogen, Carlsbad, USA), attB sites were incorporatedinto the PCR primers, and the amplified product was cloned into pDONR221vector by GATEWAY® BP recombination reaction. The resulting fragment,flanked by attL sites, was moved by the GATEWAY® LR recombinationreaction into a binary vector. The construct DNA was then used for corntransformation as described in Example 7.

Example 6b

In order to express the Rcg1 gene throughout the plant at a high level,the coding region of the gene and its terminator were placed behind thepromoter, 5′ untranslated region and an intron of a maize ubiquitin gene(Christensen et al. (1989) Plant Mol. Biol. 12:619-632; Christensen etal. (1992) Plant Mol. Biol. 18:675-689). One of skill in the art wouldbe able to select a different promoter to express the Rcg1 gene based onthe desired expression pattern. To enable cloning using the GATEWAY®cloning system, attB sites were incorporated into PCR primers that wereused to amplify the Rcg1 gene starting at 142 bp upstream of theinitiation codon and terminating 624 bp downstream of the stop codon.The amplified product was cloned into pDONR221 (Invitrogen, Carlsbad,USA) using a GATEWAY® BP recombination reaction. After cloning, theresulting Rcg1 gene was flanked by attL sites. In the final step, theRcg1 clone was moved by GATEWAY® LR recombination reaction into a vectorwhich contained the maize ubiquitin promoter, 5′ untranslated region andfirst intron of the ubiquitin gene as described by Christensen et al.(supra) followed by GATEWAY® ATTR1 and R2 sites for insertion of theRcg1 gene, behind the ubiquitin expression cassette. The vector alsocontained a marker gene suitable for corn transformation, so theresulting plasmid, carrying the chimeric gene (maize ubiquitinpromoter-ubiquitin 5′ untranslated region-ubiquitin intron 1-Rcg1), wassuitable for corn transformation as described in Example 7.

Example 7 Agrobacterium-Mediated Transformation of Maize andRegeneration of Transgenic Plants

The recombinant DNA constructs prepared in Example 6a and 6b were usedto prepare transgenic maize plants as follows.

Maize was transformed with selected polynucleotide constructs describedin Example 6a and 6b using the method of Zhao (U.S. Pat. No. 5,981,840,and PCT patent publication WO98/32326). Briefly, immature embryos wereisolated from maize and the embryos contacted with a suspension ofAgrobacterium, where the bacteria were capable of transferring thepolynucleotide construct to at least one cell of at least one of theimmature embryos (step 1: the infection step). In this step the immatureembryos were immersed in an Agrobacterium suspension for the initiationof inoculation. The embryos were co-cultured for a time with theAgrobacterium (step 2: the co-cultivation step). The immature embryoswere cultured on solid medium following the infection step. Followingthis co-cultivation period an optional “resting” step was performed. Inthis resting step, the embryos were incubated in the presence of atleast one antibiotic known to inhibit the growth of Agrobacteriumwithout the addition of a selective agent for plant transformants (step3: resting step). The immature embryos were cultured on solid mediumwith antibiotic, but without a selecting agent, for elimination ofAgrobacterium and for a resting phase for the infected cells. Next,inoculated embryos were cultured on medium containing a selective agent,and growing transformed callus was recovered (step 4: the selectionstep). The callus was then regenerated into plants (step 5: theregeneration step), and calli grown on selective medium were cultured onsolid medium to regenerate the plants.

Example 8 The Rcg1 Gene is Necessary but not Sufficient to Confer thePhenotype Associated with the Rcg1 Locus

Transgenic plants were made as described in Example 7 using theconstructs described in Examples 6a and 6b, respectively. For both thenative Rcg1 gene and the ubiquitin Rcg1 gene constructs, 30 independentevents and 10 control events transformed with the binary vector only,lacking the constructs of Example 6a or 6b, were generated.

Leaf discs of each native gene transgenic event were harvested for totalRNA isolation. RT-PCR was performed using the gene specific primersFLP111F and FLP111R set forth in SEQ ID NOS: 37 and 38. In 29 out of 30transgenic events, the expected 637 bp RT-PCR band was presentindicating expression of the native gene construct. Disease assays wereperformed in the greenhouse on the same 30 native Rcg1 transgenic eventsto determine if the plants were resistant to Cg. To accomplish this,leaf blight assays were first carried out on 5 sibling plants of eachevent using the procedures described in Example 12. A single event wasfound to show a statistically significant reduction in disease scorerelative to control plants lacking the native Rcg1 gene construct.Plants that had been subjected to the leaf blight assay were allowed todevelop two weeks post anthesis and were then further tested by Cginoculation into the first elongated stalk internode. These stalkinfection assays showed a single transgenic event expressing the nativeRcg1 transgene to be more resistant to infection by Cg when compared tocontrol plants. However, this event differed from the positive eventidentified via the leaf infection assays.

Plants transformed with the ubiquitin Rcg1 construct described inExample 6b were analyzed in a similar fashion. RT-PCR analysis showedthat 27 out of 30 transgenic events contained the expected 637 bp band,indicating expression of the ubiquitin Rcg1 construct. When leafinfection assays were performed on 5 plants from each of the 30 events,a single event was identified that showed a statistically significantreduction in disease compared to control plants. The transgenic plantswere further analyzed by stalk infection assays. Two events were foundto exhibit increased resistance to stalk rot when compared to controlplants lacking the ubiquitin Rcg1 gene. These transgenic events did notinclude the former positive event identified in the leaf blight assays.One ubiquitin Rcg1 event showed a statistically significant increase insusceptibility to anthracnose stalk rot relative to control plants.

The results of these experiments suggest a lack of efficacy with eitherthe native or ubiquitin Rcg1 construct in leaf blight or stalk infectionassays. Although by RT-PCR, each construct was found to be expressed atlevels comparable to or greater than the level of Rcg1 found inDE811ASR(BC5), only a limited number of events were found to show astatistically significant improvement in disease score relative tocontrol plants: 2 native Rcg1 T0 events and 2 ubiquitin Rcg1 T0 events.These events were separately identified by the leaf blight and stalkinfection assays. However, positive events showing increased diseaseresistance by the leaf blight assay failed to correlate with thoseidentified by the stalk infection assay. This is in contrast to theDE811ASR(BC5) positive control which shows a clear increase inresistance relative to DE811 in both leaf blight and stalk infectionassays. In addition, no correlation was observed between measureddisease scores and level of transgene expression. Indeed, one of thepositive ubiquitin Rcg1 events failed to produce a detectable RT-PCRband upon reverse transcription of total RNA and amplification of cDNAwith FLP111F and FLP111R. The lack of correlation between the differentassays for the very small number of events that appeared to showresistance, the much larger number of events that showed no resistanceand the very different results obtained with DE811 (BC5) led to thehypothesis that while the Rcg1 gene is necessary for the phenotypeconferred by the Rcg1 locus, it alone is not sufficient. The smallamount of apparent resistance observed is due to normal experimentalvariation.

In order to test the hypothesis noted above and to exclude thepossibility that errors inherent in the Rcg1 coding region of bothconstructs give rise to production of a non-functional protein that isnot competent in resistance expression, an experiment was designed thattested the ability of the transgenes to complement a transposon knockoutin Rcg1. The insertion line m164, identified in the MP305 Mu-taggedpopulation and described in Example 4, contains a transposable elementin Exon 1 of the Rcg1 gene and exhibits an anthracnose-susceptiblephenotype upon infection with Cg. This line therefore has all thecomponents of the Rcg1 locus, but the Rcg1 gene is not functional due tothe transposon insertion. Two native and two ubiquitin Rcg1 T1 lines(i.e., two each of the lines carrying the constructs of Example 6a and6b respectively) were crossed with the Rcg1 mutant line m164. Progenyfrom that cross were screened by leaf painting with glufosinate toidentify those plants containing the selectable marker gene thatco-segregates with the Rcg1 transgenes. Plants carrying the Mu-taggedRcg1 gene were identified by PCR analysis of extracted DNA using an Rcg1gene specific primer defined in SEQ ID NO: 259 and the Mu-TIR primer ofSEQ ID NO: 233. Leaf sheaths of V7-9 plants were inoculated with 5 μl ofa 5×10⁶/mL suspension of C. graminicola conidia following wounding oftissue. Inoculated leaf sheaths were sealed in plastic wrap for 5 daysto retain high moisture levels. At that time, the uppermost infectedsheath was excised for quantitation of lesion size by LemnaTec(LemnaTec, Wurselen, Germany) image analysis.

The data from the leaf sheath infection assays are summarized in FIG.33, and a typical result for one experiment is shown in FIG. 27. In theabsence of the native Rcg1 transgene, leaf sheaths of plants containingthe Mu-tagged Rcg1 gene display large, expanding necrotic lesions thattypify the reaction of this ASR susceptible plant. In contrast, when thenative Rcg1 transgene is present, an ASR resistant phenotype is restoredand necrosis is confined or restricted to the site of wounding andfungal inoculation. The data in FIG. 33 show that the necrotic areadecreases from a mean of 0.76 cm² in the absence to 0.14 cm² in thepresence of the native Rcg1 transgene. Similarly, lesion size in plantscontaining the transposon-tagged Rcg1 gene decreases from a mean valueof 0.89 cm² in the absence of the ubiquitin Rcg1 transgene to 0.11 cm²in the presence of the ubiquitin Rcg1 transgene. Thus, both the nativeand the ubiquitin Rcg1 transgenes are able to complement the knockoutmutation in Rcg1 and to restore an ASR resistant phenotype, indicatingthat both constructs are functional. The fact that neither transgenicconstruct alone is able to provide resistance indicates that the Rcg1gene is not sufficient for stalk rot resistance. The Rcg1 locustherefore contains an additional component that is required forresistance to Cg. The Rcg1 gene is therefore necessary but notsufficient.

Example 9 Analysis of DE811ASR BAC Clone Sequences to IdentifyAdditional ASR Resistance Pathway Components

The BAC clones from Example 2 covering the Rcg1 introgressed region ofDE811 ASR were examined to search for potential candidate genes that maybe required in addition to the Rcg1 gene for stalk rot resistance. Thesequences of four overlapping BAC clones were inspected to identifypredicted genes with homology to putative disease response-relatedproteins. A 79 kb supercontig (a 79 kb stretch of ordered, nearlycontinuous sequence, designated supercontig H) in BAC clone 191 wasfound to contain sequences encoding a protein showing homology toseveral NBS-LRR proteins from barley and rice. The alignment is shown inFIGS. 31 a through 31 g. This segment of the BAC contig covered 4101 bp,contained 2 predicted exons, one intron and encoded an 1164 amino acidORF (open reading frame). Comparison of the sequence against a databaseof protein signatures revealed the presence of a typical LRR domain, aNB domain with subdomains and a short, protein kinase-like sequence inthe last 37 amino acids of the ORF. In order to determine whether theNBS-LRR sequence was encoded by a functional gene that was expressed inDE811ASR plants, RT-PCR was performed. Total RNA (750 ng) fromuninoculated and Cg inoculated (3, 6, 9, 13 DPI) DE811 and DE811ASRsamples was copied in cDNA using SUPERSCRIPT® III Reverse Transcriptase(Invitrogen, Carlsbad, Calif., USA) and amplified using PLATINUM® TaqDNA Polymerase (Invitrogen, Carlsbad, Calif., USA). The cDNA synthesisreaction was assembled according to the protocol supplied by themanufacturer using random hexamers as primers and incubated at 50° C.for 50 minutes. The reaction was then terminated by heating at 85° C.for 5 minutes and the RNA template was removed by digestion with RNaseHat 37° C. for 20 minutes. Primer pairs were selected to amplify 3different fragments from the NBS-LRR cDNA template.

These PCR primer pairs consisted of ETJ111 (SEQ ID NO: 234) and ETJ112(SEQ ID NO: 235); ETJ113 (SEQ ID NO: 236) and ETJ114 (SEQ ID NO: 237);ETJ115 (SEQ ID NO: 238) and ETJ116 (SEQ ID NO: 239). The cDNA (1 μL ofthe reverse transcriptase reaction) was amplified for 30 cyclesconsisting of 30 seconds at 94° C., 30 seconds at 50° C. and 1 minute at72° C. followed by a 7 minute extension at 72° C. FIG. 28 shows theresults of RT-PCR performed with the ETJ115-ETJ116 primer pair. Thisdata clearly shows that the NBS-LRR gene of BAC clone 191 supercontig His expressed exclusively in DE811ASR plants that contain the MP305introgressed region. Similar results were obtained when cDNAamplification was carried out using the ETJ111-112 and the ETJ113-114primer pairs. DNA sequence analyses verified that all three RT-PCRproducts were derived from the NBS-LRR sequence of BAC191 supercontig H.

The NBS-LRR sequence obtained from the BAC clone did not contain anobvious stop codon and thus was only partial in nature. In order todefine the end of the coding region and to obtain a complete sequence ofthe transcript, a 3′ RACE experiment was carried out. Total RNA fromDE811ASR uninoculated and Cg inoculated (6DPI) plants was copied intocDNA using SUPERSCRIPT® III RT and an oligodT primer using standardconditions. The cDNA was then subjected to two rounds of amplificationwith PLATINUM® Taq DNA Polymerase using the nested gene specific primersoETJ118 (SEQ ID NO: 241) and oETJ119 (SEQ ID NO: 242) and an oligodTadaptor primer, oETJ117 (SEQ ID NO: 240). The results of this procedureprovided an additional 976 bp of sequence. Combination of the sequencesfrom BAC clone 191 and the 3′ RACE product leads to the prediction of anapproximate 4.5 kb transcript encoding a 1428 amino acid polypeptide. Inorder to confirm this composite sequence, a full length cDNA wasisolated by RT-PCR using SUPERSCRIPT® III RT in combination withPHUSION™ Hot Start High Fidelity DNA Polymerase (New England Biolabs,Ipswich, Mass., USA). cDNA synthesis was primed by oligodT using totalRNA from DE811ASR (0, 13 DPI) plants. Amplification with PHUSION™polymerase employed the gene specific primers oETJ 120 (SEQ ID NO: 243)and oETJ121 (SEQ ID NO: 244) which were positioned to amplify a fragmentextending from the translational start site to 31 bp beyond the stopcodon. Sequence analysis of the resultant 4318 bp fragment showedperfect agreement between the cDNA sequence and the composite BAC cloneplus RACE product sequence.

The sequence of the 4318 bp cDNA fragment is given in SEQ ID NO: 245.The deduced 1428 amino acid sequence of the 4287 bp ORF is set forth inSEQ ID NO: 246. The gene was designated Rcg1b. A BLAST®P homology searchperformed with the 1428 aa sequence revealed good homology to NBS-LRRsequences over the first ˜900 amino acids of the sequence (FIGS. 31 athrough 31 g). In addition, good homology was found between amino acids1036-1399 of the sequence (set forth in SEQ ID NO: 256) and severalputative protein kinases (FIGS. 32 a and 32 b). When the proteinsequence is queried against a database of protein families, domains andsites, a tripartite structure consisting of an NB-ARC, LRR and proteinkinase domain is obtained. The domain structure of the Rcg1b polypeptideis diagrammed in FIG. 29. The inclusion of these three domains within asingle polypeptide is not commonly found. However, a barley NBS-LRR-PKgene, Rpg5, proposed to function in resistance to Puccinia graminis hasbeen identified (Brueggeman et al., (2005) Abstracts of Plant and AnimalGenomes XIII Conference, Poster 320 [online], [retrieved on 2007-Jun.8].

Comparison of the cDNA sequence with the genomic BAC clone sequenceindicates that the Rcg1b gene is divided into 9 exons and 8 introns andcovers greater than 44 kb. Introns 2-8 are concentrated in the proteinkinase portion of the gene. Two of these introns are extremely long:intron 2 is 16.7 kb and intron 5 is 20.6 kb. The initial genomicsequence that was obtained for the Rcg1b gene and its flanking regionsis provided in SEQ ID NOs:255-258. Due to the highly repetitive natureof some of the sequence elements between the two genes, some areas ofthe genomic DNA presented significant sequencing difficulties. Afterinitial sequencing, there were three gaps of sequence located in intron5 of the Rcg1b genomic sequence. The segment of the genomic sequencecorresponding to the first 5 exons and the first 4 full introns, as wellas the start of the 5th intron (prior to the first sequence gap) is setforth in SEQ ID NO: 255. This segment includes the start codon (ATG),the TATA box, and a CAAT element. The second and third segments of thegenomic sequence of Rcg1b correspond to two portions of the 5th intron,between which the second sequence gap exists, and are set forth in SEQID NOs: 256 and 257. The fourth and final segment of the genomicsequence of Rcg1b corresponds to the remainder of the 5th intron, afterthe third sequence gap, through the remaining 3 full introns and 4 fullexons. This segment is set forth in SEQ ID NO: 258, and includes thestop codon of the Rcg1b sequence, as well as the 3′ UTR and 3′ flankinggenomic sequences. Further sequencing efforts were able to eliminate thegaps in intron 5 of the genomic sequence for Rcg1b. The complete genomicsequence for the Rcg1b gene is therefore set forth in SEQ ID NO: 266,with the positions of all exons and introns indicated in the sequencelisting accompanying this application. There is a small region withinSEQ ID NO: 266 represented by a stretch of 21 “n” residues that is not agap, but rather is a result of poor sequence quality in which the 21positions have yet to be positively confirmed. At present, the completeDNA sequence of the genomic interval between Rcg1 and Rcg1b is not knownbecause there is one remaining small gap in the genomic DNA sequence,located between the two genes, which has proven difficult to sequencebecause of repetitive elements.

Northern blot analysis allowed an independent confirmation of Rcg1bexpression and provided an estimate of transcript size. Northern blotsof polyA+ RNA (0.7 μg) from resistant and susceptible plants (0, 6, 13DPI) showed the presence of a 4.75 kb transcript band, present only inthe resistant samples that hybridized specifically with probes preparedfrom the LRR and the kinase region of the gene. (FIG. 30). The observedtranscript size agrees well with the size calculated from cDNA and RACEproducts (4.68 kb). An MPSS® tag predicted from the Rcg1b cDNA sequenceis found to be present at a low level (≦144 ppm) only in DE811ASRlibraries. This finding is consistent with RT-PCR and Northern blotresults and confirms the exclusive presence of this low abundancetranscript in ASR resistant plants.

Example 10 Dual Expression of Rcg1 and Rcg1b as Transgenes: Both Rcg1and Rcg1b Transgenes are Required for Resistance to Colletotrichumgraminicola

The Rcg1 and Rcg1b genes can be expressed as transgenes, allowingmodulation of their expression in different circumstances. The followingdisclosure shows how the two genes could be expressed in different waysto combat different diseases or to protect different portions of theplant, or simply to move the two genes into different corn lines astransgenes, as an alternative to moving the entire Rcg1 locus throughcrossing as described in Example 5.

For assembly of an Rcg1b gene driven by its native promoter, 3 Rcg1bfragments were joined together in a GATEWAY® entry vector containingattL3 and attL4 recombination sites, separated by a multiple cloningsite region. A 2.4 kb BamHI-KpnI fragment containing 1967 bp of upstreamsequence and 432 bp of the Rcg1b coding region, a 3.7 kb KpnI-BsaIfragment of the Rcg1b cDNA, and a 1.1 kb BsaI-SmaI fragment containing146 bp of the Rcg1b cDNA plus 939 bp of 3′ flanking DNA were joined in a4-way ligation to BamHI-SmaI digested L3/L4 entry vector to giveConstruct A. The native promoter driven Rcg1b construct was thentransferred by recombination, along with the native Rcg1 construct ofExample 6a into a destination vector containing attL1/attL2 andattL3/attL4 sites to give a stacked Rcg1 promoter-Rcg1/Rcg1bpromoter-Rcg1b construct (Construct B).

For high level constitutive expression, a ubiquitin promoter-drivenRcg1b construct was generated. For this construct, an Rcg1b fragmentspanning nucleotides 1 through 4318 of the cDNA sequence was amplifiedas a BamHI-SnaBI fragment by RT-PCR and inserted into a GATEWAY® entryvector containing the ubiquitin promoter, 5′UTR, intron and the PinIIterminator flanked by attL3 and attL4 sites to produce Construct C. Ananalogous construct was generated for Rcg1 (Construct D), in this casein a GATEWAY® entry vector where the ubiquitin promoter and PinIIterminator are flanked by attL1 and attL2 sites. Both the Rcg1b and Rcg1ubiquitin driven genes were transferred by an LR recombination reactioninto the destination vector noted before this time to yield a stackedUbi-Rcg1/Ubi-Rcg1b construct (Construct E). One of skill in the artwould be able to select a different promoter to express the Rcg1 andRcg1b genes, or two different promoters, each one expressing one of thetwo genes.

The above-described constructs were transformed into corn using themethod of Example 7. Two separate experiments were performed involvingtransformation of the construct from Example 6a, Construct A andConstruct B in one experiment and Construct C, Construct D and ConstructE in the other. For the first experiment, a total of 15, 10 and 30events were generated from transformation of corn with the constructfrom Example 6a, Construct A and Construct B, respectively. To confirmexpression of the DNA constructs, molecular analyses were performed.Leaf discs of each native gene transgenic event were harvested for totalRNA isolation. RT-PCR was performed using the Rcg1-specific primersAlex1F and Alex1R set forth in SEQ ID NOs: 260 and 42, respectively, andthe Rcg1b-specific primers ETJ144 and EJT145 set forth in SEQ ID NOs:261 and 262, respectively. Upon RT-PCR, all 10 of the Construct A eventscontained the expected 491 bp Rcg1b band and all 15 of the eventstransformed with the construct from Example 6a contained the expected420 bp Rcg1 band. Of the 30 events generated from transformation withthe stacked Rcg1 and Rcg1b native genes (Construct B), 29 expressed bothtransgenes while a single event was found to express one transgene(Rcg1) but not the other (Rcg1b). Disease assays were performed in thegreenhouse on the same transgenic events in order to determine whetherthe plants were resistant to Cg. Leaf sheath assays were first carriedout on 3 sibling plants of each event using the procedure described inExample 8. Infected plants were scored 5 days after inoculation with Cgby quantitation of lesion size through LemnaTec (LemnaTec, Würselen,Germany) image analysis. No significant difference in lesion size wasfound between transgenic plants containing any one of the 3 differentconstructs (Table 6a). Mean lesion size for plants containing theconstruct from Example 6a was 1.286 cm², while that for Construct A andConstruct B were 1.070 and 1.174 cm², respectively. Thus, while RT-PCRdata indicated that all three constructs showed good expression intransgenic plants, no effect on resistance to Cg could be demonstratedfor either the Rcg1 or Rcg1b native gene construct alone or for thestacked construct containing both genes in combination. Similar resultswere obtained when the same transgenic events were subjected to stalkinoculation with Cg.

TABLE 6a Transgenic Plant Results: Lesion Size DNA # of Plants MeanLesion Size (cm²) Std Deviation Construct from 34 1.286 0.683 Example 6aConstruct A 25 1.070 0.567 Construct B 78 1.174 0.530

In contrast to the results with the native gene constructs, significantdifferences in Cg resistance were found for transgenic plants containingthe constructs driven by the maize ubiquitin promoter. As shown in Table6b, mean lesion size for plants containing the single gene Construct C(Ubi-Rcg1b) and Construct E (Ubi-Rcg1) were 1.879 and 1.897 cm²,respectively. However, plants containing the stacked Ubiquitin-geneconstruct (Construct D) showed a significant reduction in lesion areawith the mean lesion size corresponding to 0.345 cm². These results areshown in FIGS. 34 through 36. RT-PCR analysis confirmed expression ofthe Rcg1b transgene in 15/15 Construct C events and the Rcg1 transgenein 13/15 Construct E events. Good expression of both Ubiquitin-promoterdriven transgenes was found in 27/30 Construct D events. Two eventslacked detectable expression of both transgenes; one event showed strongexpression of Rcg1b, but no apparent expression of Rcg1. The leaf sheathinfection assay data indicate that neither Rcg1 nor Rcg1b alone conferresistance to Cg and instead that both Rcg1 and Rcg1b are required forexpression of the resistance phenotype.

TABLE 6b Transgenic Plant Results: Lesion Size DNA # of Plants MeanLesion Size (cm²) Std Deviation ASR 8 0.480 0.259 DE811 8 4.538 0.970Construct C 38 1.879 1.097 Construct E 37 1.897 1.030 Construct D 780.345 0.650

An interesting and unexpected observation noted above is that the Rcg1and Rcg1b transgenes, driven by the ubiquitin promoter, together provideresistance to infection by Cg while the transgenes driven by theirendogenous promoter do not. It is known that the ubiquitin promoter (incombination with maize ubiquitin intron I) is able to generate highlevel constitutive expression of marker and trait genes in assortedtissues under assorted developmental and environmental conditions.Detailed characterization of the Rcg1 or Rcg1b promoter fragments usedin the constructs of the present invention has not been carried out. TheRcg1 native gene in the construct from Example 6a was shown in Example 8to complement a Mu-insertion knockout in the gene, indicating that theconstruct and thus the included promoter fragment is functional. Sinceno detailed studies of the Rcg1b promoter fragment of Construct A andConstruct B have been carried out, it appears that not all of thenecessary information required for accurate regulation of the gene isencoded in this 5′ fragment. The infection data indicate that when the1835 bp promoter fragment is used to drive expression, Rcg1b is notcorrectly expressed. Although the functional Rcg1b promoter has not yetbeen defined, the Ubi-Rcg1b results demonstrate that any functionalpromoter may be used to drive expression of the gene and this may becombined with Rcg1 expression to produce resistance to infection by Cg.

Example 11 Analysis of Rcg1 Gene Distribution Across Germplasm andIdentification of Rcg1 Sequence Variants

Following the identification, sequencing and fine mapping of Rcg1, otherlines were screened for the Rcg1 gene. To determine the presence of theRcg1 gene in other maize germplasm, gene specific primers combinationsFLP111F and FLP111R as well as FLP113F and FLPA1R were used to amplifygenomic DNA from a diverse panel of maize inbred lines, including thoselines listed on Table 18 and F2834T, by polymerase chain reaction. Inonly 14 (including MP305) out of the panel of maize inbred lines anamplification product was detected, indicating that the Rcg1 gene isonly present in a very small percentage of the inbred lines that werescreened. Thus, in addition to using MP305 or DE811ASR (BC5) as thedonor source, other sources containing the Rcg1 gene can also be used asa donor source. For example the public inbred lines TX601 (availableunder ID ‘Ames 22763’ from National Plant Germplasm System (NPGS)) andF2834T (available under ID ‘Ames 27112’ from NPGS) which contain theRcg1 gene can be used as donor sources in crosses with other maizeinbred lines not containing the Rcg1 gene, and selecting for the Rcg1gene by using markers as described herein.

Variants of the Rcg1 gene were also identified and analyzed for singlenucleotide polymorphisms (SNPs). SNPs were identified at positions onSequence ID number 1 corresponding to one or more of position 413, 958,971, 1099, 1154, 1235, 1250, 1308, 1607, 2001, 2598 and 3342. (See Table7). Not all of the allelic variants of the Rcg1 gene indicated aresistant phenotype. Therefore, these SNPs can be used as markers toprecisely identify and track the Rcg1 sequence in a plant breedingprogram, and to distinguish between resistant and susceptible allelicvariants. Further, these SNPs indicate that there are variant sequencesthat show a resistant phenotype and can be used in the methods andproducts disclosed herein. Four other lines have also been found tocontain an Rcg1 allele: BYD10, 7F11, CML261 and CML277. Testing of 10plants did not provide sufficient data to conclusively determine whetherline 7F11 is resistant. No data are available on the resistance of theBYD10, CML261 and CML277 lines.

TABLE 7 SNPs identified in allelic variants of the Rcg1 gene # PlantsConsensus position Phenotype Tested 413 958 971 1099 1154 1235 1250 13081607 2001 2598 3342 SEQ ID NO: 1 Resistant Over 500 A A G C C A A C A AG C from plants over DE811ASR 4-5 years (BC5) PHBTB Resistant150-210, over A A G C C A A : A A G C 3 years PH26T Resistant 50, over 1A A G C C A A : A A G C year TX601 Insufficient 10, over 1 A A G C C ? A: A A G C data year F2834T No data — A A G C C A A : A A G C B54 No data— C C C T A A T : G G A A PH0RC Insufficient 19, over 1 C C C T A A T :G G A A data year PH277 Insufficient 17, over 1 C C C T A A T : G G A Adata year PHDGP Susceptible 150-210, over C C C T A A T : G G A A3 years PHDH7 No data — C C C T A A T : G G A A MP305 Resistant 50 A A GC C A A C A A G C (public) Length of Consensus = 4212 nucleotides. SEQID NO: 1 is the Rcg1 sequence. For the remaining lines, the sequenceavailable spanned from the “atg” start codon in the first exon to the“tga” stop codon in the second exon. The consensus position is based onSEQ ID NO: 1.

Example 12 Lines Containing the Rcg1 Locus are Resistant toAnthracnose-Induced Leaf Blight

The near isogenic lines DE811 and DE811ASR described in Example 1 weretested for differences in resistance to leaf blight caused by Cg usingthe following procedure. Four common household sewing needles were gluedto a metal support such that the holes for the thread extended out fromthe piece of metal, with all four needles extending an equal distance.This apparatus was dipped in a suspension of Cg spores at 5×10⁶spores/mL and then pushed through the surface of a young corn leaf suchthat the leaf was wounded and the wounds simultaneously inoculated withthe spores. A wet cotton swab was placed on the midrib near theinoculation site and the entire area covered with plastic film and, overthat, reflective cloth, both attached with tape, to keep it moist andshaded. The plants were left in this state for 50-54 hours in a standardgreenhouse, after which the tape, cloth and plastic film were removed.At 7 and 15 days after inoculation the size of the lesion was measuredand recorded in units of square centimeters.

FIG. 10 (a-b) shows the distribution of lesion sizes 15 days afterinoculation across all the individual leaves. Lesion sizes vary in eachdata set, but virtually all of the DE811 leaves (FIG. 10 b) had lesionsizes significantly larger than the largest lesions to be found on theDE811ASR(BC5) leaves (FIG. 10 a). The data are summarized for both the 7day and 15 day post-inoculation data sets in FIG. 11. At both 7 and 15days, the average lesion size was smaller on the leaves carrying theRcg1 gene. The difference becomes larger over time as the fungus hastime to grow and cause further damage, so that while the difference isapproximately two fold at 7 days, by 15 days it is more than four foldand in fact the fungus has made only minor progress on the DE811ASR(BC5)leaves. These results clearly demonstrate that the presence of the locuscontaining the Rcg1 gene confers resistance to anthracnose leaf blight.

Example 13 Hybrid Lines Derived from DE811ASR(BC5) have Higher Yieldthan Hybrids Derived from DE811 when Infected with Colletotrichumgraminicola

In order to demonstrate that corn hybrids containing the Rcg1 locus havehigher yield potential when infected with Cg than hybrid lines withoutRcg1, DE811ASR (BC5) and DE811, the isogenic lines described in Example1, were each crossed to inbred lines B73Ht and Mo17Ht, which are bothsusceptible to Cg.

The hybrid lines were grown and evaluated for response to Cg in 2005 atsix locations in five different states of the USA. For each hybrid line,three replications of four rows were planted at approximately 74,000plants per hectare. Plants were inoculated with Cg at the base of thestalk approximately 10 days after flowering. The first row of eachfour-row plot was evaluated to determine if the inoculations had beensuccessful by determining the response to Cg four to five weeks afterinoculation. The stalks were split and the progression of the diseasewas scored by observation of the characteristic black color of thefungus as it grows up the stalk. Disease ratings were conducted asdescribed by Jung et al. (1994) Theoretical and Applied Genetics,89:413-418). The total number of internodes discolored greater than 75%(antgr75) was recorded on the first five internodes (See FIG. 20). Thisprovided a disease score ranging from 0 to 5, with zero indicating nointernodes more than 75% discolored and 5 indicating completediscoloration of the first five internodes. The center two plots wereharvested via combine at physiological maturity and grain yield in kg/hawas determined.

The results summarized over all locations are shown in FIG. 12 fordisease severity and in FIG. 13 for yield. The data show that hybridscontaining Rcg1 (DE811ASR(BC5)/B73Ht and DE811ASR(BC5)/Mo17Ht) have muchless disease progression than hybrids without Rcg1 (DE811/B73Ht andDE811/Mo17Ht). The high scores for disease progression in thesusceptible hybrids (lacking Rcg1) show the successful infection of theexperiment with Cg. Furthermore, the data show that when infected withCg, hybrids containing the Rcg1 locus have a higher yield than hybridslacking Rcg1. Differences of the individual pairwise comparisons aresignificant at P<0.05.

These results clearly demonstrate that by using the methods of theembodiments one can create hybrids that yield more kg of grain perhectare when infected with Cg.

Example 14 Inbred and Hybrid Rcg1 Locus Conversions Derived fromDE811ASR (BC5) or MP305 are Resistant to Colletotrichum graminicolaInduced Stalk Rot

In order to demonstrate that commercial corn lines can be made resistantto Cg-induced stalk rot, MP305 and DE811ASR (BC5) were crossed withPH87P, PH5W4, and PH705. The resulting progeny were crossed again to thesame three lines (i.e., the lines were used as recurrent parents in abackcrossing program) three more times, each time selecting for thepresence of the Rcg1 locus using molecular markers as described inExample 5 above. As controls, selected backcross lines which lacked theRcg1 locus were also collected from the same backcrossing program. Afterthree backcrosses were completed, several versions were selected andselfed to obtain BC3S1 families. Individual BC3S1 plants were genotypedand plants homozygous positive and homozygous negative for Rcg1 wereselfed to obtain BC3S2 families, which were then phenotyped. BC3S2versions containing Rcg1 and, as controls, selected versions without thegene, were planted in single row plots containing approximately 25plants per row. The experiment was planted in five different locationsin five different states of the United States, designated Locations 1,2, 3, 4, and 5. At approximately two weeks after flowering, plants wereinoculated with Cg at the base of the stalk. Four to five weeks laterthe stalks were split and progression of the disease evaluated byvisually estimating the amount of disease in the stalk. A visual scorewas assigned to each stalk based on the degree of infection of eachinternode for the inoculated internode and the four internodes above theinoculation internode. A low score thus indicates resistance to thedisease. The compiled results for all rows and locations are summarizedin FIG. 14. Representative pictures of two lines are shown in FIGS. 15and 16. The data show that at all locations, each of the elite inbredlines was made more resistant to the disease by the presence of the Rcg1locus.

Corn seed sold to farmers is “hybrid,” meaning that it is most commonlythe result of a cross of two inbred parents, referred to as a singlecross hybrid. Many years of breeding and production experience haveshown that the use of single cross hybrids result in higher yields. Itis thus important for commercial applications that the Rcg1 genefunction in the hybrid plants (those in the farmer's production field)even when it is present in only one of the two parents used to makesingle cross hybrid seed. One of the inbred lines into which the Rcg1line had been crossed, PH705, was thus used to create hybrid seed bycrossing with PH4CV, an elite inbred that does not carry the Rcg1 locus.The resulting hybrid seeds were used in experiments identical to thosedescribed for the inbred lines as discussed above and scored in the sameway at all five locations. The data are summarized for all locations inFIG. 17, which also shows the performance of the inbred PH705, andrepresentative pictures shown in FIGS. 18 and 19. As can be seen, aclear difference in disease progression was observed in all locationsfor hybrid PH705×PH5W4 and in four of the five locations forPH705×PH87P. In the fifth location, environmental conditions were verystressful for plant growth, resulting in plants that were in poorcondition. Under these conditions, measurements of plant diseaseresistance are often not reliable.

The results with both inbred lines and hybrid combinations containingRcg1 clearly demonstrate that using the methods of the embodiments onecan create commercially useful lines that are resistant to Cg-inducedstalk rot.

Example 15 Markers within the Rcg1 Coding Sequence, Marker Locations andDesigns within the Rcg1 Locust and Haplotypes for the FlankingChromosomal Region

Three levels of marker locations may be utilized as a result of the finemapping and cloning of the Rcg1 and Rcg1b genes: markers designed withinthe Rcg1 or Rcg1b coding sequences, markers designed within thenon-colinear region that identify the Rcg1 locus (but outside of theRcg1 and Rcg1b coding sequences), and markers designed within theflanking colinear region.

Following the identification and fine mapping of the Rcg1 gene,hybridization markers were designed that will function on SNP platforms.Since the Rcg1 gene occurs in a non-colinear region of the maize genome,the hybridization marker will be present in lines comprising the Rcg1gene and absent on lines that do not comprise the Rcg1 gene. Thesemarkers identify polynucleotide sequences specific to the Rcg1 codingsequence listed on SEQ ID NO: 1. As noted in Table 7, there are othercorn lines with variants of the Rcg1 coding sequence set forth in SEQ IDNO: 1, and these markers were also designed to also identify these Rcg1coding sequence variants.

To accomplish this, a consensus map of variant Rcg1 coding sequence fromdifferent sources was created, as shown on Table 7. This consensus mapaligned 4209 bases of the Rcg1 coding sequence isolated from MP305 with3451 bases from PHBTB and 3457 bases from PH26T. The Rcg1 gene in bothPHBTB and PH26T show resistance to anthracnose. Next, segments of theRcg1 coding sequence were compared using BLAST® against severaldatabases including NT (Public DNA from NCBI) and the highest homologyhits were aligned with the Rcg1 consensus sequence to determine thesegments that shared high homology and had common segments with otherresistance genes in the NBS-LRR family. Regions unique to the Rcg1coding sequence and common across the different sources of Rcg1 wereselected for marker design. Specifically, since FLP111F and FLP111Rprimers produced a single amplicon that reliably diagnosed the presenceof Rcg1 from different sources, the regions where FLP111F and FLP111Rhybridized were therefore targeted for development of a SNP markerdesign.

An INVADER™ detection system marker was designed using a 1413 bp segmentfrom the consensus sequence that contained both primer sites, with theprimer regions themselves being targeted for probe and INVADER™ oligohybridization. Primers were designed around each probe site to give anamplicon size below 150 bp. This marker indicated the presence of theRcg1 coding sequence with fluorescence due to hybridization, with theabsence of the Rcg1 coding sequence resulting in no fluorescence. Acontrol fluorescence signal can also be generated by designing a markerthat hybridizes to a second highly conserved maize gene, so that thepresence of the Rcg1 coding sequence results in fluorescence of two dyes(Rcg1 and the conserved gene) and the absence of Rcg1 results influorescence due to the conserved gene only. This ‘control’ florescencemay be used to reduce lab error by distinguishing between the situationswhere the Rcg1 is in fact absent and the situation where a falsenegative has occurred because of a failed reaction. Such markers are notlimited to a specific marker detection platform. TAQMAN® markers werealso designed to the same location (primer pairs FLP111F and FLP111R),that were used as for the INVADER™ detection system markers. The markersare shown on Table 15 and FIGS. 23 and 24.

The marker designs C00060-01-A and C00060-02-A were tested across a widevariety of sources and were highly successful at identifying plants thatcontained the Rcg1 locus and the Rcg1 gene, regardless of the source ofthe Rcg1 locus or Rcg1 gene. These markers were also used against acontrol set of nearly 100 diverse inbred lines known not to carry thegene, and no fluorescence was detected in the control set. Plants inwhich one or both of marker designs C00060-01-A and C00060-02-Aconfirmed as having Rcg1 include those shown in Table 7.

Therefore, this example shows that, based on the teaching providedherein, markers can be constructed that identify the Rcg1 codingsequence in a variety of sources.

Markers within the Rcg1 Locus

Markers may be designed to the Rcg1 locus in addition to or instead ofusing markers within the Rcg1 or Rcg1b coding sequences themselves. Theclose physical distance between the Rcg1 coding sequence and thenon-colinear region makes it unlikely that the linkage between markerswithin the non-colinear region but outside of the Rcg1 coding sequencewould be lost through recombination. As with markers for the Rcg1 codingsequence, a marker showing as present or absent would be sufficient toidentify the Rcg1 locus.

To design markers for this region, a 64,460 bp segment of non-colinearregion including the Rcg1 gene and the region directly north of the Rcg1gene (therefore between Rcg1 and Rcg1b) was initially sequenced. BACs inthis sequence were broken up into sub-clones of approximately 800nucleotides in length and sequenced. These sequences were then assembledto construct the BAC sequence, and genic and repetitive regions wereidentified. Repetitive regions were identified in order to avoid placingmarkers in repetitive regions. Similarly, sequences with high homologywith known maize sequences were easily avoided by a simple BLAST search.Potential sequences were avoided that contained SSRs, runs of As, Ts orGs, or that would result in the generation of probes low in GC contentwhich can cause problems within the INVADER™ detection system platform.See FIG. 9( b) and Table 17.

Selected segments were then put into INVADER CREATOR™ software, whichgenerates oligos for an INVADER™ reaction. This produced a sense and ananti-sense design for all SNPs. The sense designs with the best scoresand no penalties were selected. Although these markers have beendesigned, they have not yet been tested.

Primers were designed using Primer3 (Steve Rozen and Helen J. Skaletsky(2000). Primer3 is available on the world wide web for general users andfor biologist programmers. See, Krawetz S, Misener S (eds)Bioinformatics Methods and Protocols: Methods in Molecular Biology.Humana Press, Totowa, N.J., pp 365-386. Primers were selected outside ofthe INVADER™ components, and preferred primers close to or below 150 bplong were selected. Primer temperature and length was adjusted to bemost useful for the INVADER™ detection system platform, although ifusing other detection platforms primers would be optimized for use withsuch platforms.

Markers in the Colinear Region and Associated Haplotypes

Closely linked markers flanking the Rcg1 locus may be effectively usedto select for a progeny plant that has inherited the Rcg1 locus from aparent that comprises the Rcg1 locus. The markers described herein, suchas those listed on Table 16, as well as other markers genetically orphysically mapped to the same chromosomal segment, may be used to selectfor a truncated chromosomal segment comprising the Rcg1 locus.Typically, a set of these markers will be used, (e.g., 2 or more, 3 ormore, 4 or more, 5 or more) in the flanking region above the gene and asimilar set in the flanking region below the gene. Optionally, asdescribed above, a marker within the Rcg1 gene, the Rcg1b gene and/orRcg1 locus may also be used. The parents and their progeny are screenedfor these sets of markers, and the markers that are polymorphic betweenthe two parents are used for selection. The most proximal polymorphicmarkers to the Rcg1 gene, the Rcg1b gene or Rcg1 locus are used toselect for the gene or locus, and the more distal polymorphic markersare used to select against the gene or locus. In an introgressionprogram, this allows for selection of the Rcg1 gene, the Rcg1b gene orRcg1 locus genotype at the more proximal polymorphic markers, andselection for the recurrent parent genotype at the more distalpolymorphic markers. As described in more detail in Example 5 above,this process allowed for the efficient selection of a truncatedchromosomal segment comprising the Rcg1 locus.

The process described above requires knowledge of the parental genotypesused in the cross. Optionally, haplotypes may be used so that the Rcg1gene, the Rcg1b gene or Rcg1 locus can be selected for without firstgenotyping the specific parents used in the cross. This is a highlyefficient way to select for the Rcg1 locus, especially in the absence ofusing markers within the Rcg1 gene, the Rcg1b gene or the Rcg1 locus.

All plants to be used in the breeding program, such as a geneintrogression program, are screened with markers. The markers disclosedherein or equivalent markers on the same chromosomal segment may beused. The plant haplotypes (a series of SNP or other markers in linkagedisequilibrium) are noted. The haplotype of the resistant plant aroundthe Rcg1 locus is compared with the haplotype of the other plants to beused that do not comprise the Rcg1 locus. A haplotype unique to theresistant plant around the Rcg1 locus is then used for selection, andthis haplotype will specifically identify the chromosomal segment fromthe resistant plant with the Rcg1 locus.

Based on an analysis of MP305 and a diverse set of several hundred cornlines, including 50 public corn lines shown in Table 18, a unique SNPhaplotype for the MP305 chromosomal segment with the Rcg1 locus wasidentified. This SNP haplotype uniquely identifies the MP305 chromosomalsegment that extends across MZA3434, MZA2591 and MZA11123. See FIG. 22,SEQ ID NO: 140, 141 and 142, and Tables 8, 9 and 10.

First, the primer pairs described in Table 2 for these three MZA's wereused to identify haplotypes. The primer pairs MZA3434 E forward andreverse were used to amplify the genomic DNA of the set of corn lines.The PCR fragments were further purified by amplification with MZA3434 Iforward and reverse primer pairs. This process was repeated for MZA2591and MZA11123. The resulting PCR fragments were sequenced in the forwardand reverse direction and the sequences were aligned to give a consensussequence (see the sequences set forth in SEQ ID NOs: 140, 141 and 142).SNPs and indels within these consensus sequences are shown in Tables 8,9 and 10. These series of SNPs and indels were compared across the setof genotypes.

For MZA3434, haplotype 8 was a rare haplotype allele, and was unique toMP305 and only one other corn line. This process was repeated forMZA2591, and MP305 was found to have haplotype 2 at MZA2591, which wasshared by only two other corn lines. MP305 was the only corn line tohave both haplotype 8 at MZA3434 and haplotype 2 at MZA2591, andtherefore, the combination of these two haplotypes, 8 at MZA3434 and 2at MZA2591, uniquely identifies the MP305 chromosomal region comprisingthe Rcg1 locus. MP305 also had an informative haplotype at MZA11123.MP305 was found to have haplotype 7, which was shared by 66 other cornlines, but none of these corn lines had haplotype 8 at MZA3434, orhaplotype 2 at MZA2591. Therefore, any combination of 2 haplotypes atMZA3434, MZA2591 or MZA11123 could be used to uniquely identify MP305among these genotypes. The haplotypes can then be interrogated bysequencing the fragment or by designing markers to each SNP or indelwithin a fragment.

Polymorphisms within haplotypes can be used to tag the haplotype. Socalled ‘Tag-SNPs’, or ‘haplotype-tags’ can be very useful in plantbreeding, as more information than the polymorphism itself can bedetermined via extrapolation to the haplotype. A haplotype can also bedefined as a series of polymorphisms across sequences, and these may betermed ‘long-range haplotypes’.

Rare polymorphisms were observed within haplotypes that could be used as‘haplotype tags’. For example, either the SNPs MZA2591.32 (allele c) orMZA2591.35 (allele t) could be used to tag the haplotype 2 at MZA2591,and like haplotype 2, both were unique to MP305 and two other cornlines. The combination of SNPs MZA2591.32 (allele c) and MZA2591.35(allele t) combined with MZA3434.17 (allele c) gave a ‘long-range’haplotype that could be used to distinguish MP305 from all of the othergenotypes in the study.

In addition, other markers, MZA15842, MZA11455, MZA8761 and MZA1851 alsoshowed polymorphism with MP305. For MZA15842, only 18 of the other cornlines shared the same haplotype as MP305; for MZA11455, only 43 of theother corn lines shared the same haplotype as MP305; for MZA8761, onlyabout half of the other corn lines shared the same haplotype as MP305;and for MZA1851, only about half of the other corn lines shared the samehaplotype as MP305. Consensus sequences were developed for thesemarkers, and are set forth in SEQ ID NOs: 143-146. SNPs and indelswithin these consensus sequences are shown in Tables 11-14. Fourexamples of unique haplotypes using the MZA markers are:

-   MZA11123 (haplotype 7)-   MZA15842 (haplotype 3)-   MZA8761 (haplotype 1)-   and-   MZA11123 (haplotype 7)-   MZA15842 (haplotype 3)-   MZA1851 (haplotype 1)-   And-   MZA11455 (haplotype 6)-   MZA11123 (haplotype 7)-   MZA15842 (haplotype 3)-   MZA16510 (haplotype 4)-   and-   MZA11455 (haplotype 6)-   MZA11123 (haplotype 7)-   MZA15842 (haplotype 3)-   MZA11394 (haplotype 6).

Multiple combinations within all of the markers disclosed herein, orother markers within the region, also will contain unique haplotypesthat identify the Rcg1 locus.

TABLE 8 MZA3434 Polymorphisms MZA3434.3 MZA3434.4 MZA3434.6 MZA3434.17MZA3434.2 MZA3434.5 Nucleotide position 282 283 327 343 377 387 on SEQID NO: 140 Type DEL DEL DEL SNP DEL DEL Size of indel  6  1  4  2  2MP305 W M W C W M Counter allele M W M T M W M = “Mutant’: differs toconsensus W = ‘wild type’: same as consensus,

TABLE 9 MZA2591 Polymorphisms MZA2591.43 MZA2591.20 MZA2591.21 MZA2591.8MZA2591.12 MZA2591.4 MZA2591.31 MZA2591.32 Nucleotide position 101 114124 131 160 176 213 223 on SEQ ID NO: 141 Type INS SNP SNP DEL DEL INSSNP SNP Size of indel  3 2 3 MP305 W T C W W W T C Counter allele M A TM M M C T MZA2591.1 MZA2591.33 MZA2591.35 MZA2591.36 MZA2591.37MZA2591.38 MZA2591.10 MZA2591.39 Nucleotide 238 250 257 264 271 282 290310 position on SEQ ID NO: 141 Type DEL SNP SNP SNP SNP SNP DEL SNP Sizeof indel  2  4 MP305 M C T C G C M T Counter allele W G A G A T W CMZA2591.3 MZA2591.40 MZA2591.41 MZA2591.6 MZA2591.7 MZA2591.9 Nucleotideposition 313 325 332 332 371 404 on SEQ ID NO: 141 Type DEL SNP SNP DELDEL DEL Size of indel  2  1 MP305 M T C W W W Counter Allele W C T M M MM = “Mutant’: differs to consensus W = ‘wild type’: same as consensus,

TABLE 10 MZA11123 Polymorphisms MZA11123.5 MZA11123.18 MZA11123.2MZA11123.13 MZA11123.34 MZA11123.37 MZA11123.40 MZA11123.41 Nucleotide631 641 650 671 703 727 744 786 position on SEQ ID NO: 142 Type DEL INSINS INS SNP SNP SNP SNP Size of indel  1  1  1  10 MP305 W W W W G T C ACounter allele M M M M A C A G MZA11123.45 MZA11123.48 MZA11123.9MZA11123.19 MZA11123.59 MZA11123.17 MZA11123.16 Nucleotide 807 864 915934 956 991 1010 position on SEQ ID NO: 142 Type SNP SNP INS DEL SNP DELDEL Size of indel  18  1  3   3 MP305 C T W W C M W Counter allele A A MM T W M M = “Mutant’: differs to consensus W = ‘wild type’: same asconsensus,

TABLE 11 MZA15842 Polymorphisms MZA15842.3 MZA15842.4 MZA15842.5MZA15842.7 MZA15842.8 Nucleotide position 287 295 313 337 353 on SEQ IDNO: 143 Type SNP SNP SNP SNP SNP MP305 T A T C T Counter Allele C G A TC MZA15842.9 MZA15842.10 MZA15842.11 MZA15842.12 MZA15842.3 Nucleotideposition 366 436 439 463 287 on SEQ ID NO: 143 Type SNP SNP SNP SNP SNPMP305 T G A A T Counter Allele C A G G C M = “Mutant’: differs toconsensus W = ‘wild type’: same as consensus,

TABLE 12 MZA8761 Polymorphisms MZA8761.3 MZA8761.6 MZA8761.7 MZA8761.8MZA8761.9 MZA8761.10 MZA8761.11 MZA8761.4 Nucleotide 595 633 671 681 687696 702 710 position on SEQ ID NO: 145 Type DEL SNP SNP SNP SNP SNP SNPDEL Size of indel  7  1 MP305 W G T G T G C W Counter allele M A C C C TA M MZA8761.2 MZA8761.1 MZA8761.5 MZA8761.12 MZA8761.13 MZA8761.14Nucleotide position 710 710 722 779 882 901 on SEQ ID NO: 145 Type DELINS DEL SNP SNP SNP Size of indel  1  1  1 MP305 W W W T C T Counterallele M M M G T C M = “Mutant’: differs to consensus W = ‘wild type’:same as consensus,

TABLE 13 MZA1851 Polymorphisms MZA1851.24 MZA1851.41 MZA1851.32MZA1851.49 MZA1851.51 MZA1851.52 Nucleotide position 1213 1236 1271 14651615 1617 on SEQ ID NO: 144 Type INS SNP INS SNP SNP SNP Size of indel 19  34 MP305 W G W A C A Counter Allele M A M G A C MZA1851.53MZA1851.54 MZA1851.55 MZA1851.56 MZA1851.35 Nucleotide position 16861697 1698 1701 1717 on SEQ ID NO: 144 Type SNP SNP SNP SNP DEL Size ofindel   6 MP305 T A G T W Counter Allele C C C C M M = “Mutant’: differsto consensus W = ‘wild type’: same as consensus,

TABLE 14 MZA11455 Polymorphisms MZA11455.3 MZA11455.5 MZA11455.2MZA11455.7 MZA11455.8 MZA11455.10 MZA11455.11 MZA11455.12 Nucleotide 373392 402 425 426 432 435 491 position on SEQ ID NO: 146 Type DEL SNP DELSNP SNP SNP SNP SNP Size of indel  1  10 MP305 M G M G C C A T Counterallele W C W A G G G A MZA11455.4 MZA11455.13 MZA11455.14 MZA11455.15MZA11455.1 MZA11455.17 MZA11455.18 MZA11455.19 Nucleotide 526 552 581599 610 611 628 634 position on SEQ ID NO: 146 Type DEL SNP SNP SNP DELSNP SNP SNP Size of indel  1  3 MP305 M A G G W G C A Counter allele W GA C M A G C M = “Mutant’: differs to consensus W = ‘wild type’: same asconsensus,

TABLE 15 Markers within the Rcg1 Coding Sequence SNP Platform INVADER ™INVADER ™ TAQMAN ® TAQMAN ® PCR Marker Name C00060-01-A C00060-02-AC00060-01 C00060-02 FLP111 Forward Primer C00060-01-F1 C00060-02-F1C00060-01-F-Taq C00060-02-F-Taq FLP111F Name Position on 550-5671562-1586 552-568 1634-1659 595-619 SEQ ID NO: 1 Forward Primer SEQ IDNO: 145 SEQ ID NO: 146 SEQ ID NO: 147 SEQ ID NO: 148 SEQ ID NO: 37Sequence Reverse Primer C00060-01-R1 C00060-02-R1 C00060-01-R-TaqC00060-02-R-Taq FLP111RB Name Position on 641-658 1739-1767 599-6201707-1730 1676-1700 SEQ ID NO: 1 Reverse Primer SEQ ID NO: 149 SEQ IDNO: 150 SEQ ID NO: 151 SEQ ID NO: 152 SEQ ID NO: 153 Sequence Probe NameC00060-01-PCA C00060-02-PCA C00060-01-P-Taq C00060-02-P-Taq Position on586-603 1685-1701 570-595 1662-1693 SEQ ID NO: 1 Probe Sequence SEQ IDNO: 154 SEQ ID NO: 155 SEQ ID NO: 156 SEQ ID NO: 157

TABLE 16 Markers contained within defined chromosomal intervals that canbe used to select for Rcg1. Interval (and Position position on relativeto IBM2 neighbors Rcg1 or 4 map in cM) Rcg1b Markers that could be usedfor selection of Rcg1 UMC2041 Above the UMC2041, AY112127, UMC1086,AY110631 , UMC2285, (483.93)- Rcg1b gene MZA8136, MZA6064, NPI270,NPI300C, PHP20071, UMC2200 UMC2041 - CDO127a, RGPI102, UAZ122, BNL17.05,MZA11455, (543.44) Rcg1b MZA15842, MZA11123, MZA2591 Below the PHI093,MZA1215, MZA1216, MZA3434, CL12681_1, Rcg1 gene NPI444, UMC15a, MZA8761,CSU166a, CDO365, Rcg1 - CSU1038b, CSU1073b, CSU597a, RGPG111, UMN433,UMC2200 PHP20562, C2, NPI910, CSU178a, CSU202, TDA44, MZA1851, UMC1051,MZA11394, PCO136722, UMC2187, NPI410, PSR109B, UMC1371, UMC1842,UMC1856, AY109980, UMC1132, NFD106, AY105971, AY110989, ENSI002A,RZ596B, BNL23A, BNL29, UMC2200 UMC1086 Above the UMC1086, AY110631,UMC2285, MZA8136, MZA6064, (500.59)- Rcg1b gene NPI270, NPI300C,PHP20071, CDO127a, RGPI102, UMC2200 UMC1086 - UAZ122, BNL17.05,MZA11455, MZA15842, MZA11123, (543.44) Rcg1b MZA2591 Below the PHI093,MZA1215, MZA1216, MZA3434, CL12681_1, Rcg1 gene NPI444, UMC15a, MZA8761,CSU166a, CDO365, Rcg1 - CSU1038b, CSU1073b, CSU597a, RGPG111, UMN433,UMC2200 PHP20562, C2, NPI910, CSU178a, CSU202, TDA44, MZA1851, UMC1051,MZA11394, PCO136722, UMC2187, NPI410, PSR109B, UMC1371, UMC1842,UMC1856, AY109980, UMC1132, NFD106, AY105971, AY110989, ENSI002A,RZ596B, BNL23A, BNL29, UMC2200 UMC2285 Above the UMC2285, MZA8136,MZA6064, NPI270, NPI300C, (514.9)- Rcg1b gene PHP20071, CDO127a,RGPI102, UAZ122, BNL17.05, UMC2187 UMC2285 - MZA11455, MZA15842,MZA11123, MZA2591 (531.7) Rcg1b Below the PHI093, MZA1215, MZA1216,MZA3434, CL12681_1, Rcg1 gene NPI444, UMC15a, MZA8761, CSU166a, CDO365,Rcg1 - CSU1038b, CSU1073b, CSU597a, RGPG111, UMN433, UMC2187 PHP20562,C2, NPI910, CSU178a, CSU202, TDA44, MZA1851, UMC1051, MZA11394,PCO136722, UMC2187 Within Above the MZA8136, MZA6064, NPI270, NPI300C,PHP20071, UMC2285 Rcg1b gene, CDO127a, RGPI102, UAZ122, BNL17.05,MZA11455, (514.9)- within MZA15842, MZA11123, MZA2591 UMC15a UMC2285 -(525.8) Rcg1b Below the PHI093, MZA1215, MZA1216, MZA3434, CL12681_1,Rcg1 gene, NPI444 within Rcg1 - UMC15a The public markers are taken fromthe IBM2 neighbors 4 map, while the relative locations of the Pioneermarkers (prefix ‘MZA’) were determined by mapping to the same geneticmap, and by location on the physical map.

TABLE 17 Markers Within the Rcg1 Locus SNP sequence Marker position onINVADER ™ INVADER ™ Name SEQ ID NO: 137 SNP Sequence Oligo Probe ForwardPrimer Reverse Primer PHD0001-01  12-270 SEQ ID NO: 158 SEQ ID NO: 159SEQ ID NO: 160 SEQ ID NO: 161 SEQ ID NO: 162 PHD0002-01 272-530 SEQ IDNO: 163 SEQ ID NO: 164 SEQ ID NO: 165 SEQ ID NO: 166 SEQ ID NO: 167PHD0003-01 7232-7500 SEQ ID NO: 168 SEQ ID NO: 169 SEQ ID NO: 170 SEQ IDNO: 171 SEQ ID NO: 172 PHD0004-01 11302-11580 SEQ ID NO: 173 SEQ ID NO:174 SEQ ID NO: 175 SEQ ID NO: 176 SEQ ID NO: 177 PHD0005-01 11581-11880SEQ ID NO: 178 SEQ ID NO: 179 SEQ ID NO: 180 SEQ ID NO: 181 SEQ ID NO:182 PHD0006-01 11881-12170 SEQ ID NO: 183 SEQ ID NO: 184 SEQ ID NO: 185SEQ ID NO: 186 SEQ ID NO: 187 PHD0007-01 12171-12470 SEQ ID NO: 188 SEQID NO: 189 SEQ ID NO: 190 SEQ ID NO: 191 SEQ ID NO: 192 PHD0008-0125417-25690 SEQ ID NO: 193 SEQ ID NO: 194 SEQ ID NO: 195 SEQ ID NO: 196SEQ ID NO: 197 PHD0009-01 25692-25950 SEQ ID NO: 198 SEQ ID NO: 199 SEQID NO: 200 SEQ ID NO: 201 SEQ ID NO: 202 PHD0010-01 25951-26200 SEQ IDNO: 203 SEQ ID NO: 204 SEQ ID NO: 205 SEQ ID NO: 206 SEQ ID NO: 207PHD0011-01 26602-26860 SEQ ID NO: 208 SEQ ID NO: 209 SEQ ID NO: 210 SEQID NO: 211 SEQ ID NO: 212 PHD0012-01 26932-27200 SEQ ID NO: 213 SEQ IDNO: 214 SEQ ID NO: 215 SEQ ID NO: 216 SEQ ID NO: 217 PHD0013-0127322-27580 SEQ ID NO: 218 SEQ ID NO: 219 SEQ ID NO: 220 SEQ ID NO: 221SEQ ID NO: 222 PHD0014-01 28472-28740 SEQ ID NO: 223 SEQ ID NO: 224 SEQID NO: 225 SEQ ID NO: 226 SEQ ID NO: 227 PHD0015-01 28791-29002 SEQ IDNO: 228 SEQ ID NO: 229 SEQ ID NO: 230 SEQ ID NO: 231 SEQ ID NO: 232

TABLE 18 List of Public Lines use in Haplotype Analysis 38-11 A165 A188A509 A556 A619 A632 B B14 B37 B42 B64 B73 B84 B89 B94 C103 C106 CI66CM49 CO109 D02 D146 F2 F252 F257 F283 F7 GT119 H84 H99 HATO4 HY IndianaH60 K187-11217 K55 L1546 L317 Minn49 MO13 MP305 N28 OH07 OH40B OH43 OH45OS420 OS426 PA91 R159 SC213R SD105 SRS303 T232 TR9-1-1-6 TX601 V3 W153RWF9

1. An isolated polynucleotide comprising a polynucleotide selected fromthe group consisting of: (a) a nucleotide sequence encoding apolypeptide capable of conferring or enhancing resistance toColletotrichum when co-expressed in a plant with a second nucleotidesequence encoding SEQ ID NO: 3, wherein the polypeptide has an aminoacid sequence of at least 95% identity, when compared to SEQ ID NO: 246based on GAP version 10 under default parameters; (b) a complement ofthe nucleotide sequence of (a), wherein the complement and thenucleotide sequence consist of the same number of nucleotides and are100% complementary; and (c) a nucleotide sequence encoding a polypeptideconferring or enhancing resistance to Colletotrichum, when co-expressedin a plant with a second nucleotide sequence encoding SEQ ID NO: 3,wherein the polypeptide has an amino acid sequence of at least 95%identity, when compared to the sequence of the plasmid deposited asPatent Deposit No. NRRL B-50050 based on GAP version 10 under defaultparameters.
 2. The polynucleotide of claim 1, wherein the nucleotidesequence comprises SEQ ID NO:
 245. 3. The polynucleotide of claim 1,wherein the nucleotide sequence comprises the sequence of the plasmiddeposited as Patent Deposit No. NRRL B-50050.
 4. A vector comprising thepolynucleotide of claim
 1. 5. A recombinant DNA construct comprising thepolynucleotide of claim 1 operably linked to at least one regulatorysequence.
 6. The recombinant DNA construct of claim 5 further comprisinga second polynucleotide encoding the Rcg1 peptide set forth in SEQ IDNO:
 3. 7. A plant cell comprising the recombinant DNA construct of claim5 or claim
 6. 8. A process for producing a plant comprising transforminga plant cell with the recombinant DNA construct of claim 5 or claim 6and regenerating a plant from the transformed plant cell.
 9. A plantcomprising the recombinant DNA construct of claim 5 or claim
 6. 10. Theplant of claim 9, wherein said plant is maize.
 11. A seed comprising therecombinant DNA construct of claim 5 or claim
 6. 12. The seed of claim11, wherein said seed is maize seed.
 13. A process of conferring orenhancing resistance to Colletotrichum, comprising transforming a plantwith the recombinant DNA construct of claim 5 or claim 6, therebyconferring or enhancing resistance to Colletotrichum as compared to anon-transformed plant.
 14. A process of altering the level of expressionof proteins capable of conferring resistance to Colletotrichum in aplant cell comprising: (a) transforming a plant cell with therecombinant DNA construct of claim 6; and (b) growing the transformedplant cell under conditions that are suitable for expression of therecombinant DNA construct wherein expression of the recombinant DNAconstruct results in production of altered levels of proteins capable ofconferring resistance to Colletotrichum in the transformed plant cell.15. A process of altering the level of expression of proteins capable ofconferring resistance to stalk rot in a plant cell comprising: (a)transforming a plant cell with the recombinant DNA construct of claim 6;and (b) growing the transformed plant cell under conditions that aresuitable for expression of the recombinant DNA construct whereinexpression of the recombinant DNA construct results in production ofaltered levels of proteins capable of conferring resistance to stalk rotin the transformed plant cell.
 16. A process of altering the level ofexpression of proteins capable of conferring resistance toColletotrichum in a plant comprising: (a) transforming a plant cell withthe recombinant DNA construct of claim 6; and (b) regenerating atransformed plant from the transformed plant cell; and (c) growing thetransformed plant under conditions that are suitable for expression ofthe recombinant DNA construct wherein expression of the recombinant DNAconstruct results in production of altered levels of proteins capable ofconferring resistance to Colletotrichum in the transformed plant.
 17. Aprocess of altering the level of expression of proteins capable ofconferring resistance to stalk rot in a plant comprising: (a)transforming a plant cell with the recombinant DNA construct of claim 6;and (b) regenerating the transformed plant from the transformed plantcell; and (c) growing the transformed plant under conditions that aresuitable for expression of the recombinant DNA construct whereinexpression of the recombinant DNA construct results in production ofaltered levels of proteins capable of conferring resistance to stalk rotin the transformed plant.